CN115531297A - Injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and preparation method and application thereof - Google Patents
Injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and preparation method and application thereof Download PDFInfo
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- CN115531297A CN115531297A CN202211333558.2A CN202211333558A CN115531297A CN 115531297 A CN115531297 A CN 115531297A CN 202211333558 A CN202211333558 A CN 202211333558A CN 115531297 A CN115531297 A CN 115531297A
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Abstract
The invention relates to an injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and a preparation method and application thereof. Wherein, the three-dimensional porous structure of the microsphere provides space and support for the growth of the human umbilical cord mesenchymal stem cell ball to form the human umbilical cord mesenchymal stem cell ball; moreover, the human umbilical cord blood platelet-rich plasma contains various growth factors, so that the inflammation level can be inhibited, and the differentiation of human umbilical cord mesenchymal stem cells to cartilage is promoted, thereby improving the pathological microenvironment of articular cartilage injury and promoting the repair of articular cartilage injury. The injectable hydrogel system has good clinical application practice and prospect, and can provide a new idea and way for the treatment and recovery of articular cartilage injury.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to an injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell balls, and a preparation method and application thereof.
Background
Articular cartilage is an important tissue bearing mechanical load of human body, and is often damaged due to trauma, osteoarthritis and the like, and the quality of life of patients is seriously affected by clinical symptoms and degenerative diseases. However, since the articular cartilage itself lacks blood, lymph, nerves and undifferentiated cells which can proliferate and migrate, and the proliferation capacity of the chondrocytes itself is limited, the articular cartilage is difficult to self-heal once damaged, and the inflammatory microenvironment under pathological conditions aggravates the articular cartilage damage. At present, the problems of difficult cartilage regeneration and the like cannot be fundamentally solved by clinical conventional treatment. Due to the limited self-repair capacity of articular cartilage, replacement therapy by in vitro implanted stem cells is a promising approach and has been extensively tested clinically. However, the simple implantation of stem cells is still disadvantageous to maintain the phenotype of stem cells, make the distribution of cells uneven and the like due to the self anatomical structure of articular cartilage, and the damaged inflammatory pathological microenvironment affects the performance of stem cells, even leads to damage, death and the like of the stem cells and the stem cells. To solve the existing problems of the therapy, the invention considers that a bracket is added to provide a stable growth environment for the proliferation and the growth of cells, and growth factors are added to play the roles of resisting inflammation and promoting chondrogenesis.
The invention is intended to use the above solutions of the tissue engineering design which have been developed well in recent years. The key point of repairing the articular cartilage through tissue engineering is that proper seed cells, cell factors and cell scaffolds are selected, and the cell scaffold material needs to provide a growth environment close to the extracellular matrix of the articular cartilage for the seed cells. In tissue engineering, chondrocytes are usually transplanted at first, but the chondrocytes have the characteristics of easy aging, dedifferentiation and the like, and the long-term treatment effect of articular cartilage damage is difficult to achieve. The hydrogel has a hydrophilic three-dimensional network structure, is similar to a soft tissue due to the characteristics of a cross-linking network, namely the higher cross-linking degree and the lower water absorption amount, and is listed as a potential cartilage matrix substitute material for a long time. Due to the technical condition limitation of biomedicine in the past, the function of the traditional hydrogel material in repairing joint injury cannot be effectively played, and the development of a novel hydrogel material and the application of the novel hydrogel material in repairing cartilage injury become one of the research hotspots in the field. The hydrogel has high water content, good elasticity and certain mechanical strength, is easy to load drugs and cells, can simulate extracellular matrix, provides a proper microenvironment for cartilage injury repair, and has a good development prospect. However, most of the existing synthetic hydrogel materials have the problems of insufficient stability, biological safety and cell adhesion. Based on the above, the development of seed cells and cytokines in a suitable form, safe and effective, and capable of being used in vivo, is the key to the treatment of cartilage damage, recovery and regeneration. Therefore, the invention provides an injectable hydrogel system capable of loading platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, which can inhibit inflammation and promote directional differentiation of human umbilical cord mesenchymal stem cells so as to promote recovery of articular cartilage damage.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides an injectable hydrogel system for loading platelet-rich plasma and umbilical cord mesenchymal stem cell balls and a preparation method thereof, wherein the injectable hydrogel system is used as a porous framework with good biocompatibility, can well load human umbilical cord blood platelet-rich plasma and human umbilical cord mesenchymal stem cell balls, and has high biological safety; and the injectable hydrogel loaded with human umbilical cord blood platelet-rich plasma and human umbilical cord mesenchymal stem cell spheres can inhibit inflammation and promote the directional differentiation of human umbilical cord mesenchymal stem cells, thereby promoting the recovery of articular cartilage injury.
The purpose of the invention is realized by the following technical scheme:
an injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls comprises hydrogel, human umbilical cord blood platelet rich plasma and human umbilical cord mesenchymal stem cell balls.
Further, the hydrogel comprises sodium alginate, calcium gluconate and calcium chloride, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 15-25 and is (0.5-2).
Furthermore, each milliliter of hydrogel contains 50 to 100 microliters of human umbilical cord blood platelet-rich plasma.
Further, the platelet concentration in the human umbilical cord blood platelet-rich plasma is 1000 x 10 9 /L。
Furthermore, each milliliter of hydrogel contains 1-5 mg of human umbilical cord mesenchymal stem cell balls.
Further, the human umbilical cord mesenchymal stem cell spheres comprise microspheres and human umbilical cord mesenchymal stem cells, wherein each 1-5 mg of the microspheres contains 10 ten thousand of human umbilical cord mesenchymal stem cells.
A preparation method of the injectable hydrogel system loaded with the platelet-rich plasma and umbilical cord mesenchymal stem cell balls comprises the following preparation steps:
step one, dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a proportion at room temperature, standing for 12-24 h, then fully mixing the three solutions according to the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride of 15-25;
step two, adding human umbilical cord blood platelet-rich plasma into the hydrogel: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the first step according to the proportion of adding 50-100 microliters of human umbilical cord blood platelet-rich plasma into each milliliter of hydrogel to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 1-5 g of gelatin in 10-15 ml of ultrapure water to prepare a gelatin solution; (2) Adding 1-2g of Span80 into 80-120 ml of liquid paraffin, uniformly stirring at the temperature of 40-50 ℃, adding a prepared gelatin solution, continuously stirring, observing to generate gelatin microspheres, washing with acetone, crosslinking with 10-15% of glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand of human umbilical cord mesenchymal stem cells into each 1-5 mg of microspheres, and culturing for 22-26 h by using a stem cell ball culture medium to obtain the human umbilical cord mesenchymal stem cell ball;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and at room temperature, adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 1-5 mg of human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution, mixing, and stirring uniformly to obtain the injectable hydrogel system.
Further, in the step one, the ratio of the sodium alginate to the ultrapure water is that 15-25 mg of sodium alginate is added into each 1 ml of the ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 3-5 mg of calcium gluconate is added into 1 ml of ultrapure water; the proportion of the calcium chloride and the ultrapure water is that 1-2 mg of the calcium chloride is added into 1 ml of the ultrapure water.
Further, in step three, the stem cell pellet medium is MEM- α stem cell pellet medium containing 10% fbs and 1% diabody.
In the invention, the articular cartilage is repaired and regenerated mainly by the performance of stem cells, and the stem cells are effectively assisted by adding a scaffold and growth factors. The injectable hydrogel and the microspheres prepared by the invention have high biological safety. The microspheres can provide a three-dimensional culture system, imitate the specificity of a microenvironment in the cell growth process, provide a good three-dimensional environment similar to the microenvironment for in vivo growth for cells due to the porous structure of the microspheres, enable the cells to stably grow, and be used for culturing human umbilical cord mesenchymal stem cells to form human umbilical cord mesenchymal stem cell spheres; sodium alginate in the injectable hydrogel has the advantages of high water content, similarity with natural extracellular matrix, irregular shape formation for better filling defects and the like, and can well load umbilical cord mesenchymal stem cells and growth factors when used as a porous framework with good biocompatibility. In addition, the human umbilical cord blood platelet-rich plasma containing various growth factors is added into the bracket, and the human umbilical cord blood platelet-rich plasma containing various growth factors can play a role in resisting inflammation and promoting the directional differentiation of human umbilical cord mesenchymal stem cells. Therefore, the invention prepares an injectable hydrogel system capable of loading platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and the injectable hydrogel system can inhibit inflammation and promote the directional differentiation of human umbilical cord mesenchymal stem cells so as to promote the recovery of articular cartilage damage.
The invention provides application of the injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell balls in preparation of a therapeutic drug for treating articular cartilage damage. The articular cartilage damage includes but is not limited to traumatic arthritis, degenerative osteoarthritis, rheumatoid arthritis, tendon repair, meniscal replacement and the like. Experiments prove that the injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell balls can obviously inhibit the inflammation level and the directional differentiation of human umbilical cord mesenchymal stem cell balls, further improve inflammation and promote cartilage synthesis.
The invention has the beneficial effects that: (1) The invention provides an injectable hydrogel system for loading platelet-rich plasma and umbilical cord mesenchymal stem cell balls, which can well load human umbilical cord platelet-rich plasma and human umbilical cord mesenchymal stem cell balls and has good biocompatibility.
(2) The invention provides an injectable hydrogel system capable of loading platelet-rich plasma and umbilical cord mesenchymal stem cell balls, which can be used for treating articular cartilage damage.
(3) The invention provides an injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, wherein microspheres can provide a good three-dimensional space for human umbilical cord mesenchymal stem cells to form the human umbilical cord mesenchymal stem cell spheres, so that the survival and proliferation of the human umbilical cord mesenchymal stem cells can be promoted, and the microspheres are differentiated into cartilage spheres under the influence of the human umbilical cord platelet-rich plasma.
(5) The invention provides a preparation method of an injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, which has the characteristics of simple method, low production cost, no need of high temperature, no need of special complex and expensive equipment, and suitability for large-scale production.
(6) The injectable hydrogel system provided by the invention is used for loading human umbilical cord blood platelet-rich plasma and human umbilical cord mesenchymal stem cell balls, and can be further applied to the application of articular cartilage injury medicines through the effects of inhibiting inflammation and promoting the directional differentiation of human umbilical cord mesenchymal stem cells. Because the injectable hydrogel system is adopted to load the human umbilical cord blood platelet-rich plasma and the human umbilical cord mesenchymal stem cell balls, the three-dimensional porous structure of the microspheres can be utilized to provide a three-dimensional space and a certain mechanical support for the growth of the human umbilical cord mesenchymal stem cells; meanwhile, the three-dimensional porous structure of the injectable hydrogel is utilized to play a role of a bracket, and irregular gaps in articular cartilage injury are filled by the three-dimensional porous structure; in addition, the injectable hydrogel can be used for loading and releasing human umbilical cord blood platelet-rich plasma, so that the inflammation level is reduced, the local microenvironment is improved, and the directional differentiation of human umbilical cord mesenchymal stem cells is promoted. In conclusion, the injectable hydrogel system has good clinical application practice and prospects, and can provide new ideas and ways for treatment and recovery of articular cartilage injury.
Drawings
FIG. 1 is a schematic diagram of an injectable hydrogel system of the present invention loaded with human umbilical cord blood platelet rich plasma and human umbilical cord mesenchymal stem cell balls.
FIG. 2 is an electron micrograph of a hydrogel.
FIG. 3 is an electron micrograph of microspheres.
FIG. 4 is a graph showing the results of staining human umbilical cord mesenchymal stem cell spheres.
Detailed Description
The present invention will be further described with reference to the following examples for facilitating understanding of those skilled in the art, and the description of the embodiments is not intended to limit the present invention.
Example 1
The embodiment provides an injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell spheres, which comprises hydrogel, human umbilical cord platelet rich plasma and human umbilical cord mesenchymal stem cell spheres.
Further, the hydrogel comprises sodium alginate, calcium gluconate, calcium chloride and ultrapure water, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 15.
Further, 50 microliters of human umbilical cord blood platelet-rich plasma was contained per milliliter of hydrogel, and the platelet concentration in the human umbilical cord blood platelet-rich plasma was 1000 × 10 9 /L。
Further, each milliliter of hydrogel contains 2mg of human umbilical cord mesenchymal stem cell balls.
Further, the human umbilical cord mesenchymal stem cell sphere comprises microspheres and human umbilical cord mesenchymal stem cells, wherein each 2mg of the microspheres contains 10 ten thousand of the human umbilical cord mesenchymal stem cells.
The preparation method of the injectable hydrogel system of this embodiment includes the following preparation steps:
step one, dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a proportion at room temperature, standing for 12h, then fully mixing the three obtained solutions according to the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride of 15;
step two, adding human umbilical cord blood platelet-rich plasma into the hydrogel: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the step one according to a ratio to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 1g of gelatin in 10 ml of ultrapure water to prepare a gelatin solution; (2) Adding 2g of Span80 into 100 ml of liquid paraffin, stirring uniformly at the temperature of 40 ℃, adding a prepared gelatin solution into the liquid paraffin, continuously stirring, observing to generate gelatin microspheres, washing with acetone, crosslinking with 10% glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand of human umbilical cord mesenchymal stem cells into each 2mg of microspheres, and culturing for 22h by using a stem cell ball culture medium to obtain the human umbilical cord mesenchymal stem cell balls;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and at room temperature, adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 2mg of human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution, mixing, and uniformly stirring to obtain the injectable hydrogel system.
Further, in the step one, the ratio of the sodium alginate to the ultrapure water is that 15mg of sodium alginate is added into each 1 ml of the ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 3.5mg of the calcium gluconate is added into 1 ml of the ultrapure water; the ratio of calcium chloride to ultrapure water was 1.2mg of calcium chloride per 1 ml of ultrapure water.
Further, in step three, the stem cell pellet medium is MEM- α stem cell pellet medium containing 10% fbs and 1% diabody.
Example 2
The embodiment provides an injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell spheres, which comprises hydrogel, human umbilical cord platelet rich plasma and human umbilical cord mesenchymal stem cell spheres.
Further, the hydrogel comprises sodium alginate, calcium gluconate, calcium chloride and ultrapure water, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 17.
Furthermore, 80 microliters of human umbilical cord blood platelet-rich plasma was contained in each milliliter of hydrogel, and the platelet concentration in the human umbilical cord blood platelet-rich plasma was 1000 × 10 9 /L。
Further, each milliliter of hydrogel contains 4mg of human umbilical cord mesenchymal stem cell balls.
Further, the human umbilical cord mesenchymal stem cell ball comprises microspheres and human umbilical cord mesenchymal stem cells. Wherein, each 4mg microsphere contains 10 ten thousand of human umbilical cord mesenchymal stem cells.
The preparation method of the injectable hydrogel system of this embodiment includes the following steps:
step one, dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a proportion at room temperature, standing for 16h, then fully mixing the three solutions according to the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride of 17;
step two, adding human umbilical cord blood platelet-rich plasma into the hydrogel: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the first step according to the formula proportion to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 1g of gelatin in 15 ml of ultrapure water to prepare a gelatin solution; (2) Adding 1.5g of Span80 into 100 ml of liquid paraffin, uniformly stirring at the temperature of 42 ℃, adding a prepared gelatin solution into the liquid paraffin, continuously stirring, observing to generate gelatin microspheres, washing with acetone, crosslinking with 10% glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand of human umbilical cord mesenchymal stem cells into each 4mg of microspheres, and culturing for 24h by using a stem cell ball culture medium to obtain the human umbilical cord mesenchymal stem cell balls;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and (4) adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 4mg of the human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution at room temperature, mixing, and stirring uniformly to obtain the injectable hydrogel system.
Further, in the step one, the ratio of the sodium alginate to the ultrapure water is that 17mg of sodium alginate is added into each 1 ml of the ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 4mg of calcium gluconate is added into 1 ml of the ultrapure water; the ratio of calcium chloride to ultrapure water was 2mg of calcium chloride per 1 ml of ultrapure water.
Further, in the third step, the stem cell pellet medium is MEM-. Alpha.stem cell pellet medium containing 10% FBS and 1% diabody.
Example 3
The injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell balls provided by the embodiment comprises hydrogel, human umbilical cord platelet-rich plasma and human umbilical cord mesenchymal stem cell balls.
Further, the hydrogel comprises sodium alginate, calcium gluconate, calcium chloride and ultrapure water, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 25.
Further, 100 microliters of human umbilical cord blood platelet-rich plasma was contained in each milliliter of the hydrogel, and the platelet concentration in the human umbilical cord blood platelet-rich plasma was 1000 × 10 9 /L。
Further, 5mg of human umbilical cord mesenchymal stem cell balls are contained in each milliliter of hydrogel.
Further, the human umbilical cord mesenchymal stem cell ball comprises microspheres and human umbilical cord mesenchymal stem cells. Wherein, each 5mg microsphere contains 10 ten thousand human umbilical cord mesenchymal stem cells.
The preparation method of the injectable hydrogel system of this embodiment includes the following steps:
step one, dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a certain proportion at room temperature, standing for 20h, then fully mixing the three obtained standing solutions according to the mass ratio of 25;
step two, adding human umbilical cord blood platelet-rich plasma into the hydrogel: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the first step to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 5g of gelatin in 15 ml of ultrapure water to prepare a gelatin solution; (2) Adding 2g of Span80 into 120 ml of liquid paraffin, stirring uniformly at the temperature of 45 ℃, adding a prepared gelatin solution into the liquid paraffin, continuously stirring, observing to generate gelatin microspheres, washing with acetone, crosslinking with 15% glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand of human umbilical cord mesenchymal stem cells into each 5mg of microspheres, and culturing for 26h by using a stem cell ball culture medium to obtain the human umbilical cord mesenchymal stem cell balls;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and (4) at room temperature, adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 5mg of human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution, mixing, and stirring uniformly to obtain the injectable hydrogel system.
Further, in the step one, the ratio of the sodium alginate to the ultrapure water is that 25mg of sodium alginate is added into each 1 ml of the ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 3mg of calcium gluconate is added into each 1 ml of ultrapure water; the ratio of calcium chloride to ultrapure water was 1mg of calcium chloride per 1 ml of ultrapure water.
Further, in the third step, the stem cell pellet medium is MEM-. Alpha.stem cell pellet medium containing 10% FBS and 1% diabody.
Example 4
The injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell balls provided by the embodiment comprises hydrogel, human umbilical cord platelet-rich plasma and human umbilical cord mesenchymal stem cell balls.
Further, the hydrogel comprises sodium alginate, calcium gluconate, calcium chloride and ultrapure water, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 20.
Further, the hydrogel contained 75 microliters of human umbilical cord blood platelet rich plasma per milliliter, wherein the platelet concentration in the human umbilical cord blood platelet rich plasma was 1000 x 10 9 /L。
Further, each milliliter of hydrogel contains 3mg of human umbilical cord mesenchymal stem cell balls.
Further, the human umbilical cord mesenchymal stem cell ball comprises microspheres and human umbilical cord mesenchymal stem cells. Wherein, each 3mg microsphere contains 10 ten thousand of human umbilical cord mesenchymal stem cells.
The preparation method of the injectable hydrogel system of this embodiment includes the following steps:
dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a certain proportion at room temperature, standing for 24h, then fully mixing the three obtained standing solutions according to the mass ratio of 20;
step two, adding human umbilical cord blood platelet-rich plasma into the hydrogel: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the step one according to a formula ratio to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 1g of gelatin in 15 ml of ultrapure water to prepare a gelatin solution; (2) Adding 1g of Span80 into 90 ml of liquid paraffin, stirring uniformly at the temperature of 40 ℃, adding a prepared gelatin solution into the liquid paraffin, continuously stirring, observing to generate gelatin microspheres, washing the gelatin microspheres with acetone, crosslinking with 12% glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand of human umbilical cord mesenchymal stem cells into each 3mg of microspheres, and culturing for 24h by using a stem cell ball culture medium to obtain the human umbilical cord mesenchymal stem cell balls;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and at room temperature, adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 3mg of human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution, mixing, and uniformly stirring to obtain the injectable hydrogel system.
Further, in the step one, the ratio of the sodium alginate to the ultrapure water is that 20mg of sodium alginate is added into each 1 ml of the ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 5mg of calcium gluconate is added into 1 ml of the ultrapure water; the ratio of calcium chloride to ultrapure water was 0.5mg of calcium chloride per 1 ml of ultrapure water.
Further, in step three, the stem cell pellet medium is MEM- α stem cell pellet medium containing 10% fbs and 1% diabody.
Example 5
The injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell balls provided by the embodiment comprises hydrogel, human umbilical cord platelet-rich plasma and human umbilical cord mesenchymal stem cell balls.
Further, the hydrogel comprises sodium alginate, calcium gluconate, calcium chloride and ultrapure water, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 23.
Furthermore, 90 microliters of human umbilical cord blood platelet-rich plasma was contained per milliliter of hydrogel. The platelet concentration of the human umbilical cord blood platelet-rich plasma is 1000 x 10 9 /L。
Further, 1mg of human umbilical cord mesenchymal stem cell spheres per milliliter of hydrogel is contained.
Further, the human umbilical cord mesenchymal stem cell ball comprises microspheres and human umbilical cord mesenchymal stem cells. Wherein, each 1mg microsphere contains 10 ten thousand human umbilical cord mesenchymal stem cells.
The preparation method of the injectable hydrogel system of this embodiment includes the following steps:
step one, dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a certain proportion at room temperature, standing for 20h, then fully mixing the three obtained standing solutions according to the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride of 23;
step two, adding human umbilical cord blood into the hydrogelPlatelet rich plasma: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the first step to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 4g of gelatin in 15 ml of ultrapure water to prepare a gelatin solution; (2) Adding 2g of Span80 into 80 ml of liquid paraffin, uniformly stirring at 45 ℃, adding a prepared gelatin solution, continuously stirring, observing to generate gelatin microspheres, washing with acetone, crosslinking with 10% of glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand human umbilical cord mesenchymal stem cells into each 1mg of microspheres, and culturing for 26h by using a stem cell ball culture medium to obtain human umbilical cord mesenchymal stem cell balls;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and (4) at room temperature, adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 1mg of human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution, mixing, and stirring uniformly to obtain the injectable hydrogel system.
Further, in the step one, the ratio of sodium alginate to ultrapure water is 18mg per 1 ml of ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 3mg of calcium gluconate is added into 1 ml of the ultrapure water; the ratio of calcium chloride to ultrapure water was 1.5mg of calcium chloride per 1 ml of ultrapure water.
Further, in step three, the stem cell pellet medium is MEM- α stem cell pellet medium containing 10% fbs and 1% diabody.
Example 6
The injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell balls provided by the embodiment comprises hydrogel, human umbilical cord platelet-rich plasma and human umbilical cord mesenchymal stem cell balls.
Further, the hydrogel comprises sodium alginate, calcium gluconate, calcium chloride and ultrapure water, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 25.
Further, 60 microliters of human umbilical cord blood platelet rich plasma was contained per milliliter of hydrogel.
Further, the platelet concentration in the human umbilical cord blood platelet-rich plasma is 1000 x 10 9 /L。
Further, 2.5mg of human umbilical cord mesenchymal stem cell balls are contained in each milliliter of hydrogel.
Further, the human umbilical cord mesenchymal stem cell ball comprises microspheres and human umbilical cord mesenchymal stem cells.
Further, in the human umbilical cord mesenchymal stem cell sphere, 10 ten thousand human umbilical cord mesenchymal stem cells are contained in each 2.5mg of microspheres.
The preparation method of the injectable hydrogel system of this embodiment includes the following preparation steps:
step one, dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a certain proportion at room temperature, standing for 18h, then fully mixing the three obtained standing solutions according to the mass ratio of 25;
step two, adding human umbilical cord blood platelet-rich plasma into the hydrogel: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the first step to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 1g of gelatin in 10 ml of ultrapure water to prepare a gelatin solution; (2) Adding 2g of Span80 into 100 ml of liquid paraffin, stirring uniformly at the temperature of 50 ℃, adding a prepared gelatin solution into the liquid paraffin, continuously stirring, observing to generate gelatin microspheres, washing with acetone, crosslinking with 15% glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand of human umbilical cord mesenchymal stem cells into each 2.5mg of microspheres, and culturing for 24 hours by using a stem cell ball culture medium to obtain human umbilical cord mesenchymal stem cell balls;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and at room temperature, adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 2.5mg of human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution, mixing, and stirring uniformly to obtain the injectable hydrogel system.
Further, in the step one, the ratio of the sodium alginate to the ultrapure water is that 25mg of sodium alginate is added into each 1 ml of the ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 5mg of calcium gluconate is added into each 1 ml of ultrapure water; the ratio of calcium chloride to ultrapure water was 2mg of calcium chloride per 1 ml of ultrapure water.
Further, in step three, the stem cell pellet medium is MEM- α stem cell pellet medium containing 10% fbs and 1% diabody.
Example 7
An injectable hydrogel system is loaded with human umbilical cord blood platelet-rich plasma and human umbilical cord mesenchymal stem cells, and the specific operation method is as follows:
1. isolation and culture identification of seed cells:
(1) Taking and culturing human umbilical cord mesenchymal stem cells: the human umbilical cord is obtained through approval and consent of an ethical committee, and the parturient agrees and signs an informed consent. Human umbilical cord mesenchymal stem cells were isolated from umbilical cords discarded after birth of a newborn using a tissue adherence method. Clamping an umbilical cord into a culture dish by using forceps, rinsing bloodstain, shearing the umbilical cord into smaller segments, conveniently processing the segments section by section in the culture dish, extruding residual blood or blood clots in the umbilical cord, rinsing the umbilical cord, tearing off an outer amniotic membrane and an inner membrane of the umbilical cord, pulling out two umbilical arteries, discarding the remaining gelatinous part, transferring the remaining gelatinous part into a dry culture dish, and shearing the gelatinous part into 10-25 mm 2 Adding 10% of FBS-and 1% of diaquan-containing MEM-alpha complete medium to the tissue mass, and 5% CO at 37% 2 And (5) incubating the incubator.
(2) The culture method comprises the following steps: and observing the cells under a microscope, wherein the human umbilical cord mesenchymal stem cells grow in an adherent manner and are in a long fusiform shape, and under the normal condition, the cells are changed every two days and are passaged every three days. At 37 ℃ C, 5% CO 2 The incubator is used for incubation, and 3-5 generations of cells are transferred for transplantation.
(3) Identifying cultured human umbilical cord mesenchymal stem cells: detecting whether the immunophenotype of the cells meets the standard of the human umbilical cord mesenchymal stem cells by using a flow cytometer, and respectively detecting the positive expression rate of the human umbilical cord mesenchymal stem cell positive marker and the positive expression rate of the negative marker. The result shows that the cultured cells are determined to be neural stem cells.
2. Preparing and culturing human umbilical cord mesenchymal stem cell balls:
(1) Preparing microspheres: dissolving 1g of gelatin in 10 ml of ultrapure water to prepare a gelatin solution, adding 2g of Span80 into 100 ml of liquid paraffin, uniformly stirring in a constant-temperature water bath kettle at 50 ℃, adding the prepared gelatin solution, continuously stirring, observing to generate gelatin microspheres, washing with acetone to remove the liquid paraffin and the Span80 on the surfaces of the gelatin microspheres, crosslinking with 15% of glutaraldehyde, washing with ultrapure water to remove residual glutaraldehyde, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres for later use.
(2) Preparation of microspheres: and (2) putting the microspheres obtained in the step (1) into a low-adhesion 24-hole plate, and adding 500 microliters of PBS to soak the microspheres for half an hour.
(3) Preparing a suspension of human umbilical cord mesenchymal stem cells: and (3) in a biological safety cabinet or a super clean bench, absorbing and discarding the culture medium in a culture bottle, adding 1 ml of pancreatin containing EDTA with the concentration of 0.25%, digesting at 37 ℃ for 1 minute, then adding 1 ml of complete culture medium to stop digestion, collecting cells into a 15 ml centrifuge tube, centrifuging at 1200rpm for 5 minutes, and removing the supernatant to obtain the human umbilical cord mesenchymal stem cells.
(4) Preparing and culturing a human umbilical cord mesenchymal stem cell ball: removing PBS from the well plate, adding 1 ml of a cell suspension containing 10 ten thousand human umbilical cord mesenchymal stem cells per well, at 37 ℃ C. 5% 2 Incubating in incubator for 24 hr cultured cellsAfter transplantation.
3. Extracting and identifying human umbilical cord blood platelet-rich plasma:
(1) Extraction of human umbilical cord blood platelet-rich plasma: the human cord blood was obtained by approval and consent of the ethical committee, giving maternal consent and signing informed consent. Cord blood is obtained from umbilical cord discarded after birth of a newborn, and the human umbilical cord blood platelet-rich plasma is extracted by adopting a twice centrifugation method. The blood was first centrifuged at 1500rpm for 10 minutes and separated into three layers: an upper serum layer, a lower red blood cell layer and a white membrane layer (containing platelets and white blood cells) between the upper serum layer and the lower red blood cell layer. The red blood cells below 2 mm below the buffy coat were aspirated by syringe and discarded. The remaining part was centrifuged for a second time at 3000rpm for 10 minutes, after which 3/4 of the serum layer was aspirated by a syringe and discarded, and the remaining part was collected.
(2) Identification of human umbilical cord blood platelet-rich plasma: collecting the liquid, and performing platelet count with PLT not less than 1000 × 10 9 the/L represents the extraction success. The results showed that the extracted liquid was indeed human umbilical cord platelet rich plasma. Addition of PBS adjusted PLT to 1000 x 10 in human cord blood platelet rich plasma 9 /L。
4. Preparation of injectable hydrogel systems:
(1) Preparation of hydrogel: at room temperature, weighing sodium alginate, calcium gluconate and calcium chloride according to the formula, respectively adding into ultrapure water, continuously standing for 24h to dissolve the three to obtain solutions respectively, mixing the three according to the formula, and standing at 2 deg.C for 4h to obtain semitransparent liquid.
(2) Loading human umbilical cord blood platelet-rich plasma and human umbilical cord mesenchymal stem cell spheres: and (3) sucking 1 ml of hydrogel at room temperature, adding 50 microliters of human umbilical cord blood platelet-rich plasma, adding 5mg of human umbilical cord mesenchymal stem cell balls, and fully mixing to obtain the injectable hydrogel system loaded with the platelet-rich plasma and the umbilical cord mesenchymal stem cell balls for testing.
5. The results of the effect observation of the injectable hydrogel system loaded with human umbilical cord blood platelet-rich plasma and human umbilical cord mesenchymal stem cell spheres are as follows:
TABLE 1 characterization test results (X. + -. S) for hydrogels and microspheres
| Hydrogels | Microspheres | |
| Pore size of mum | 22.52±7.44 | 13.03±5.84 |
| Porosity% | 38.52±6.44 | 91.15±1.17 |
As shown in fig. 2-3, it can be known by combining the data in table 1 that both the hydrogel and the microsphere are loose and porous materials, the pore size of the microsphere is close to the diameter of the human umbilical cord mesenchymal stem cells, the porosity is large, the size is proper, and the porous microsphere can allow a large amount of cells to adhere and grow to form a stem cell sphere, so that the retention rate of the cells can be improved, and the interaction of the cells can be promoted; the hydrogel has larger pore diameter than the stem cell ball, can load the stem cell ball to play a role in fixing, and simultaneously, the hydrogel contains nearly 95 percent of water, and can slowly release the human umbilical cord blood platelet-rich plasma.
TABLE 2MTT detection of human umbilical cord mesenchymal stem cell survival rate (X + -S) after 3 days of culture of hydrogel leaching solutions of different concentrations
Note: the control group was cultured using normal medium, and the experimental group was cultured using medium containing hydrogel extract at various concentrations (1, 1/2, 1/4, 1/8, 1/16 and 1/32, respectively).
As can be seen from Table 2, the survival rates of the human umbilical cord mesenchymal stem cells of the experimental groups were more than 70%, and the cell survival rates of two experimental groups (1/16 and 1/32) were more than that of the control group, thereby indicating that the hydrogel is safe and non-toxic to the human umbilical cord mesenchymal stem cells and can promote cell proliferation.
TABLE 3 MTT detection of human umbilical cord mesenchymal stem cell viability (X + -S) after 3 days of culture of microsphere leach solutions of different concentrations
Note: the control group was cultured in normal medium, and the experimental group was cultured in medium containing different concentrations of the leaching solution of microspheres (1, 1/2, 1/4, 1/8, 1/16 and 1/32, respectively).
As can be seen from Table 3, the survival rates of the human umbilical cord mesenchymal stem cells of the experimental groups are more than 85%, and the cell survival rates of the two groups (with the concentration of 1/16 and 1/32) are more than that of the control group, thereby demonstrating that the microspheres are safe and nontoxic to the human umbilical cord mesenchymal stem cells and can promote cell proliferation.
TABLE 4 CCK-8 detection of cell viability after 3 days of culturing human umbilical cord mesenchymal stem cells in different ways
| Control group | System group | |
| Cell viability% | 100 | 133.00±3.34 |
Note: the control group is cultured in normal culture medium, the system group is cultured in culture medium containing PRP and microsphere, and the comparison between the control group and the system group is less than 0.05.
As can be seen from table 4, the cell survival rate of the system group is significantly higher than that of the control group, which indicates that the injectable hydrogel system loaded with human umbilical cord mesenchymal stem cells can significantly improve the survival rate of neural stem cells.
Example 8
An injectable hydrogel system experiment for inhibiting inflammation and promoting directional differentiation of human umbilical cord mesenchymal stem cells comprises the following specific operation methods:
1. construction and detection of in vitro inflammation model:
(1) And (3) carrying out isolated culture and identification on chondrocytes: SD rats of 3-4 weeks old are sacrificed by intraperitoneal injection of excess anesthetic. Taking cartilage on joint surface with surgical blade under aseptic operation, washing with 1% double antibody-containing PBS for several times, transferring into super clean bench, and cutting cartilage tissue to 1mm 3 And then cultured with 2 mg/ml collagenase II at 37 ℃ for 4 hours, filtered through a 200-mesh stainless steel mesh, collected cells, centrifuged, and inoculated into DMEM/F12 medium containing 1% double antibody and 10% FBS. P3 generation cells were taken for toluidine blue identification. The results showed that the extracted cells were indeed chondrocytes.
(2) Co-culturing the chondrocytes and the human umbilical cord mesenchymal stem cell spheres: the experiment was carried out using a coculture chamber, the lower layer was inoculated with 20 ten thousand chondrocytes, 2 ml of DMEM/F12 complete medium containing 20ng of proinflammatory factor IL-1. Beta. Was added to each well, 1mg of human umbilical cord mesenchymal stem cell spheres was inoculated to the upper chamber, 1 ml of MEM-alpha complete medium containing 50. Mu.l of human umbilical cord blood platelet-rich plasma was added, chondrocytes were cultured in a control group normal medium, chondrocytes were cultured in an inflammation group in 2 ml of DMEM/F12 complete medium containing 20ng of proinflammatory factor IL-1. Beta. And the plates were incubated at 37 ℃ and 5 CO 2 The culture box is used for incubation,after 24 hours of incubation, detection was performed.
(3) Detecting the level of inflammation and expression of markers specific for cartilage degradation of chondrocytes: the total RNA of the chondrocytes was extracted using Trizol kit according to the instructions. The purity and the content of RNA are measured by a micro ultraviolet spectrophotometer, and each RNA sample (A260/A280) is 1.7-2.1. The RNA was transcribed into cDNA according to the reverse transcription kit instructions. And carrying out real-time quantitative PCR detection by using the qRT-PCR kit. Taking 1 mu L of the reversed cDNA product as a template, taking GAPD hours as an internal reference, and detecting the relative expression quantity of the type II collagen gene in different treatment groups by using SYBR Green I dye real-time quantitative PCR, wherein the qRT-PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s;95 ℃ for 5s,60 ℃ for 30s,40 cycles; 15s at 90 ℃;60 ℃ for 1min. The experiment was repeated 3 times, and the average was taken and GADP hours was used as the internal control. Data analysis was performed by the Δ Δ Ct method, and data was averaged over 3 replicates.
2. Detecting the directional differentiation of the human umbilical cord mesenchymal stem cells in vitro:
(1) Cell culture: under aseptic conditions, 10 ten thousand of human umbilical cord mesenchymal stem cells (control group) and 1mg of human umbilical cord mesenchymal stem cell balls (system group) were inoculated into a well plate, 2 ml of chondrogenic induction medium was added to each well of the control group, 100. Mu.l of human umbilical cord blood platelet-rich plasma was added to the system group based on 2 ml of chondrogenic induction medium, and the plate was incubated at 37 ℃ and 5% CO 2 And (5) incubating in an incubator, and detecting after culturing for 10-14 days.
(2) Detecting the expression of the cartilage-specific markers: extracting the total RNA of the human umbilical cord mesenchymal stem cells by using a Trizol kit according to the instruction. The purity and the content of RNA are measured by a micro ultraviolet spectrophotometer, and each RNA sample (A260/A280) is 1.7-2.1. The RNA was transcribed into cDNA according to the reverse transcription kit instructions. And carrying out real-time quantitative PCR detection by using the qRT-PCR kit. Taking 1 mu L of the inverted cDNA product as a template, taking GAPD hours as an internal reference, and detecting the relative expression quantity of the type II collagen in different treatment groups by using SYBR Green I dye real-time quantitative PCR, wherein the qRT-PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s;95 ℃ for 5s,60 ℃ for 30s,40 cycles; 15s at 90 ℃;60 ℃ for 1min. The experiment was repeated 3 times, and the average was taken and GADP hour was used as the internal control. Data analysis was performed by the Δ Δ Ct method, and data was averaged over 3 replicates.
3. The results of the observation of the effects of the injectable hydrogel system in inhibiting inflammation and promoting the directional differentiation of human umbilical cord mesenchymal stem cells are as follows:
TABLE 5 qRT-PCR detection of Gene expression (X. + -. S) after 24 hours of culturing human umbilical cord mesenchymal Stem cells in different ways
| Control group | Inflammation group | System group | |
| Relative content of inflammatory factor IL-6 | 1 | 1.73±0.33 | 0.02±0.00 |
| Relative content of cartilage degradation gene MMP-13 | 1 | 1.24±0.54 | 0.12±0.03 |
Note: the inflammatory group compared to the systemic group had P < 0.05.
As can be seen from table 5, the human umbilical cord mesenchymal stem cell balls and human umbilical cord blood platelet-rich plasma loaded in the injectable hydrogel system can significantly inhibit the inflammation level and simultaneously inhibit the degradation of cartilage, thereby improving inflammation and promoting cartilage synthesis.
TABLE 6 qRT-PCR detection of Gene expression (X. + -. S) 10 days after culturing human umbilical cord mesenchymal Stem cells in different ways
| Control group | System group | |
| Relative content of cartilage marker COL II | 1 | 53.65±3.35 |
Note: the control group compared with the system group had P < 0.05.
As can be seen from Table 6, the human umbilical cord mesenchymal stem cell spheres and the human umbilical cord blood platelet-rich plasma loaded in the injectable hydrogel system can obviously inhibit and promote the directional differentiation of the human umbilical cord mesenchymal stem cells.
The above specific examples are further illustrative of the technical solutions and advantages of the present invention, and are not intended to limit the embodiments. It will be apparent to those skilled in the art that any obvious alternative is within the scope of the invention without departing from the inventive concept.
Claims (10)
1. An injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls, which is characterized in that: comprises hydrogel, human umbilical cord blood platelet-rich plasma and human umbilical cord mesenchymal stem cell balls.
2. The injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls according to claim 1, wherein: the hydrogel comprises sodium alginate, calcium gluconate and calcium chloride, wherein the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride is 15-25.
3. The injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls according to claim 2, wherein: each milliliter of hydrogel contains 50-100 microliter of human umbilical cord blood platelet-rich plasma.
4. The injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls according to claim 3, wherein: the platelet concentration in the human umbilical cord blood platelet-rich plasma is 1000 x 10 9 /L。
5. The injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls according to claim 2, wherein: each milliliter of hydrogel contains 1-5 mg of human umbilical cord mesenchymal stem cell spheres.
6. The injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls according to claim 5, wherein: the human umbilical cord mesenchymal stem cell ball comprises microspheres and human umbilical cord mesenchymal stem cells, wherein each 1-5 mg of the microspheres contains 10 ten thousand of the human umbilical cord mesenchymal stem cells.
7. A method of preparing an injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell spheres as claimed in any one of claims 1 to 6, wherein: the preparation method comprises the following preparation steps:
dissolving and mixing hydrogel: respectively dissolving sodium alginate, calcium gluconate and calcium chloride in ultrapure water according to a proportion at room temperature, standing for 12-24 h, then fully mixing the three solutions according to the mass ratio of the sodium alginate to the calcium gluconate to the calcium chloride of 15-25;
step two, adding human umbilical cord blood platelet-rich plasma into the hydrogel: PBS was added to adjust platelet concentration in human cord blood platelet rich plasma to 1000 x 10 9 At room temperature, mixing and stirring the human umbilical cord blood platelet-rich plasma and the semitransparent liquid substance obtained in the first step according to the proportion of adding 50-100 microliters of human umbilical cord blood platelet-rich plasma into each milliliter of hydrogel to obtain a second mixed solution;
step three, preparing the microspheres and the human umbilical cord mesenchymal stem cell spheres: (1) Dissolving 1-5 g of gelatin in 10-15 ml of ultrapure water to prepare a gelatin solution; (2) Adding 1-2g of Span80 into 80-120 ml of liquid paraffin, uniformly stirring at the temperature of 40-50 ℃, adding a prepared gelatin solution, continuously stirring, observing to generate gelatin microspheres, washing with acetone, crosslinking with 10-15% of glutaraldehyde, washing with ultrapure water, quickly freezing with liquid nitrogen, and freeze-drying with a freeze dryer to obtain the microspheres; (3) Adding 10 ten thousand of human umbilical cord mesenchymal stem cells into each 1-5 mg of microspheres, and culturing for 22-26 h by using a stem cell ball culture medium to obtain the human umbilical cord mesenchymal stem cell ball;
step four, adding the human umbilical cord mesenchymal stem cell ball into the hydrogel: and at room temperature, adding the human umbilical cord mesenchymal stem cell balls obtained in the third step into the second mixed solution obtained in the second step according to the proportion of adding 1-5 mg of human umbilical cord mesenchymal stem cell balls into each milliliter of the second mixed solution, mixing, and stirring uniformly to obtain the injectable hydrogel system.
8. The preparation method of the injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls according to claim 7, wherein the preparation method comprises the following steps: in the step one, the ratio of the sodium alginate to the ultrapure water is that 15-25 mg of sodium alginate is added into each 1 ml of the ultrapure water; the proportion of the calcium gluconate to the ultrapure water is that 3-5 mg of calcium gluconate is added into 1 ml of the ultrapure water; the proportion of the calcium chloride and the ultrapure water is that 1-2 mg of the calcium chloride is added into 1 ml of the ultrapure water.
9. The preparation method of the injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell balls according to claim 7, wherein the preparation method comprises the following steps: in step three, the stem cell sphere medium was MEM-alpha stem cell sphere medium containing 10% FBS and 1% diabody.
10. Use of the injectable hydrogel system loaded with platelet rich plasma and umbilical cord mesenchymal stem cell spheres of any one of claims 1 to 6 for the preparation of a medicament for the treatment of articular cartilage damage.
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