阅读说明：本技术 一种水凝胶及其制备方法与应用 (Hydrogel and preparation method and application thereof ) 是由 邹石泉 邢鹤琳 李曼 武鹏 裴振华 于 2021-09-02 设计创作，主要内容包括：本发明提供了一种水凝胶及其制备方法与应用,属于生物组织工程技术领域。本发明水凝胶,包括凝胶基质,以及干细胞和外泌体,所述干细胞经缺氧处理,所述外泌体溶于含有生长因子的培养基进行电穿孔处理。本发明提供的水凝胶能够显著提升植入细胞的存活能力,为加快损伤修复提供了可能,外泌体作为无生命的囊泡,为细胞提供了存活需要的营养和存活信号,同时其被细胞内吞后其空间也为细胞生长、迁移等提供了可能。本发明水凝胶可应用于制备修复骨缺损的产品中,在老年、糖尿病和极速修复中有广泛应用前途。(The invention provides a hydrogel and a preparation method and application thereof, belonging to the technical field of biological tissue engineering. The hydrogel comprises a gel matrix, stem cells and exosomes, wherein the stem cells are subjected to hypoxia treatment, and the exosomes are dissolved in a culture medium containing growth factors and subjected to electroporation treatment. The hydrogel provided by the invention can obviously improve the survival capability of implanted cells, provides possibility for accelerating injury repair, provides nutrition and survival signals required by survival for the cells by taking exosomes as non-living vesicles, and provides possibility for cell growth, cell migration and the like by taking the exosomes as spaces after the exosomes are endocytosed by the cells. The hydrogel can be applied to the preparation of products for repairing bone defects, and has wide application prospects in the fields of old people, diabetes and rapid repair.)

1. A hydrogel, comprising a gel matrix, stem cells that have been subjected to hypoxic treatment, and exosomes dissolved in a growth factor-containing medium for electroporation treatment.

2. The hydrogel of claim 1, wherein the gel matrix is methacrylic anhydrified gelatin.

3. The hydrogel of claim 1, wherein the hypoxic culture condition of the hypoxic treatment is 1% O₂，5％CO₂，94％N₂。

4. The hydrogel according to claim 1, wherein the culture medium comprises: 5-10 × DMEM, 150-250ng/ml PDGF, 150-250ng/ml bFGF and 5-10 × B27.

5. The hydrogel of claim 1, wherein the voltage of the electroporation is 600-800V, the capacitance is 100-200mF, the electrode spacing of the electroporation cuvette is 4mm, and the number of times is 2-4.

6. The hydrogel according to claim 1, wherein the ratio of the amount of stem cells to exosomes is 1: 10⁴-10⁵。

7. A method for preparing the hydrogel according to any one of claims 1 to 6, comprising the steps of: and mixing the stem cells and the exosomes in a culture medium, mixing the obtained mixed solution with the gel matrix, adding a photoinitiator, and inducing gelling.

8. The method according to claim 7, wherein the medium is a DMEM medium.

9. The method according to claim 8, wherein the volume ratio of the mixture to the gel matrix is 1:3 to 2: 1.

10. Use of a hydrogel according to any one of claims 1 to 6 for the preparation of a product for repairing a bone defect.

Technical Field

The invention belongs to the technical field of biological tissue engineering, and particularly relates to hydrogel and a preparation method and application thereof.

Background

Bone damage caused by trauma, infection, tumor or congenital genetic disease is a common orthopedic disorder. Bone tissue itself has a certain capacity of automatic repair and regeneration, but large areas of bone defects need to be treated by means of external intervention, and regenerative repair thereof remains a major challenge in clinical medicine. The jaw face defect is a common disease, and the main reasons for the jaw face defect comprise congenital and acquired factors: it is mainly caused by cleft lip and palate, congenital developmental deformity and trauma, jaw bone tumor or other diseases. The main effects on the human body are difficulties in chewing, aesthetic problems and speech dysfunction. The maxillofacial defect is a defect of maxillofacial parts and organs thereof including bones and soft tissues, mainly including jawbone, facial soft tissues and facial organs, and some defects can be shaped by surgery, but in many cases, the method is still used for repairing oral cavities. The primary means of repairing maxillofacial bone defects is repair plasty, and the repair of large and complex maxillofacial bone defects remains a challenge for maxillofacial surgery. The autologous bone graft material is limited by a donor and is difficult to obtain the shape matched with the bone defect part, so that the preparation of the personalized bone scaffold material aiming at the specific bone defect part has important significance.

The scaffold material is one of the key elements of bone tissue engineering, and finding a scaffold material which has good biocompatibility, better osteogenesis promoting capability and biodegradability is a hotspot and difficulty of research. The hydrogel material has the bionic characteristic of extracellular matrix and a high three-dimensional hydration network structure, and is beneficial to the migration and growth of cells in the processes of tissue regeneration and wound healing. The addition of cells into hydrogel can better promote the repair of tissue damage, but after the cells are implanted into the hydrogel, the defect of low cell survival rate exists, and the use effect of hydrogel materials is influenced.

Disclosure of Invention

In view of the above, the present invention aims to provide a hydrogel capable of significantly enhancing the survival rate of implanted cells and the bone regeneration potential.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a hydrogel which comprises a gel matrix, stem cells and exosomes, wherein the stem cells are subjected to hypoxia treatment, and the exosomes are dissolved in a culture medium containing growth factors and subjected to electroporation treatment.

Preferably, the gel matrix is methacrylic anhydrified gelatin.

Preferably, the hypoxic culture conditions of the hypoxic treatment are 1% O₂，5％CO₂，94％N₂。

Preferably, the culture medium comprises: 5-10 × DMEM, 150-250ng/ml PDGF, 150-250ng/ml bFGF and 5-10 × B27.

Preferably, the voltage of the electroporation is 600-800V, the capacitance is 100-200mF, the electrode spacing of the electroporation cup is 4mm, and the times are 2-4.

Preferably, the ratio of the dosage of the stem cells to the dosage of the exosomes is 1: 10⁴-10⁵。

The invention also provides a preparation method of the hydrogel, which comprises the following steps: and mixing the stem cells and the exosomes in a culture medium, mixing the obtained mixed solution with the gel matrix, adding a photoinitiator, and inducing gelling.

Preferably, the medium is a DMEM medium.

Preferably, the volume ratio of the mixed solution to the gel matrix is 1:3-2: 1.

The invention also provides application of the hydrogel in preparation of a product for repairing bone defects.

The invention has the beneficial effects that:

in the hydrogel, exosomes are used as non-living vesicles, and after the vesicles are subjected to engineering treatment, high-concentration nutrient components required by cell survival are loaded, so that nutrition and survival signals required by cell survival are provided for the cells, and space of the exosomes after being endocytosed by the cells also provides possibility for cell growth, cell migration and the like. The stem cells have stronger hypoxia tolerance capability through hypoxia treatment. The hydrogel designed by the invention can obviously improve the survival capability and bone regeneration potential of implanted cells, and provides possibility for accelerating bone injury repair. Has wide application prospect in the elderly, diabetes and rapid repair. The hydrogel can be combined with a 3D bioprinting technology (bio-3DP), and the hydrogel scaffold with an ideal complex three-dimensional structure is printed in a bio-3DP manner, so that the possibility of repairing the ultimate bone defect by using the high-strength hydrogel is provided.

Drawings

FIG. 1 shows the relative number of viable cells in hydrogels treated differently.

Detailed Description

The invention provides a hydrogel which comprises a gel matrix, stem cells and exosomes, wherein the stem cells are subjected to hypoxia treatment, and the exosomes are dissolved in a culture medium containing growth factors and subjected to electroporation treatment.

In the invention, the gel matrix is preferably methacrylic acid anhydrified gelatin (GelMA), wherein GelMA is a photosensitive biological hydrogel material, has excellent biocompatibility, can be excited by ultraviolet light or visible light to carry out curing reaction, and forms a three-dimensional structure which is suitable for cell growth and differentiation and has certain strength.

The type of stem cells in the present invention is not particularly limited, and any type of stem cells may be used as long as they can participate in bone regeneration. In a particular embodiment of the invention, mesenchymal stem cells are used. In the present invention, the hypoxic treatment is preferably performed by subjecting the stem cells to hypoxic culture in a low-sugar medium, wherein the hypoxic culture condition is preferably 1% O₂，5％CO₂，94％N₂The low sugar culture refers to culture using a medium having a sugar content lower than that of a general stem cell medium, and in a specific embodiment of the present invention, DMEM medium having a sugar content of 5.6mmol/L is used. In the present invention, the anoxic treatmentThe time period is preferably 5 to 9 days, more preferably 6 to 8 days. The invention carries out hypoxia treatment on the stem cells, so that the stem cells have stronger hypoxia tolerance and are more suitable for the microenvironment in the hydrogel.

The source of the exosome is not particularly required, and the exosome can be obtained by adopting an exosome obtaining way known in the field. The type of the growth factor is not particularly limited in the present invention, as long as it can enrich the exosome with the key growth factor required for the growth and survival of the cell, in the present invention, the culture medium preferably comprises 5-10 × DMEM, 150-250ng/ml PDGF, 150-250ng/ml bFGF and 5-10 × B27, more preferably comprises 6-9 × DMEM, 180-220ng/ml PDGF, 180-220ng/ml bFGF and 6-9 × B27, wherein B27 refers to an antioxidant and serum-free medium additive component, which is a common additive for neuron culture media. In the present invention, when the exosome dissolved in the growth factor-containing medium is subjected to electroporation treatment, the voltage of the electroporation is preferably 600-800V, more preferably 650-750V, the capacitance of the electroporation is preferably 100-200mF, more preferably 130-170mF, the electrode spacing of the electroporation cuvette is preferably 4mm, and the number of the electroporation is preferably 2-4, more preferably 3. The exosomes are engineered by the method of the invention to be enriched in key growth factors required for nutrition and cell growth and survival.

In the hydrogel of the present invention, the ratio of the amount of the stem cells to the exosomes is preferably 1: 10⁴-10⁵。

The invention also provides a preparation method of the hydrogel, which comprises the following steps: and mixing the stem cells and the exosomes in a culture medium, mixing the obtained mixed solution with the gel matrix, adding a photoinitiator, and inducing gelling.

In the preparation method of the present invention, the culture medium for mixing the stem cells and the exosomes is preferably a DMEM culture medium, and the specific formula of the DMEM is not particularly limited, and may be a DMEM culture medium formula conventional in the art. In the present invention, the volume ratio of the mixed solution to the gel matrix is preferably 1:3 to 2:1, more preferably 1: 1. the photoinitiator is not particularly limited in the invention, and any initiator conventional in the art can be used, in the specific embodiment of the invention, the photoinitiator is LAP (lithium phenyl-2, 4, 6-trimethylbenzoylphosphite) which is crosslinked more rapidly and has less damage to cells, and the addition amount of the LAP photoinitiator is preferably 0.5% (w/v). The method for inducing gelling is not particularly limited, in the specific embodiment of the invention, blue light is selected for inducing gelling, the wavelength of inducing gelling is preferably 400nm, and the time for inducing gelling is preferably 10S.

The invention also provides application of the hydrogel in preparation of a product for repairing bone defects.

The present invention is not particularly limited in the kind of product, and includes medical drugs and the like.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Placing Mesenchymal Stem Cells (MSC) in commercial low-sugar DMEM medium, and maintaining low-oxygen condition 1% O₂，5％CO₂，94％N₂And culturing for 7 days to make the culture more suitable for the microenvironment in the hydrogel, thereby obtaining the pretreated MSC.

Collecting plasma exosomes, dissolving in 10 × DMEM +200ng/ml PDGF +200ng/mLbFGF +10 × B27 culture medium, and performing electroporation treatment for 3 times under the conditions that the voltage is 700V, the capacitance is 150mF and the electrode spacing of an electric shocking cup is 4mm, so that the exosomes are rich in nutrition and key growth factors required by cell growth and survival, and the engineered exosomes are obtained.

And (3) performing pretreatment on the MSC and the engineered exosome according to the cell number of the MSC: exosome number was 1: 10⁴The components are mixed in a DMEM medium to obtain a mixed solution, the mixed solution is mixed with 20% gelMA (model GM-60, Intelligent manufacturing institute of Suzhou, Suzhou) in equal volume, 0.5% (w/v) LAP photoinitiator is added, and blue light with the wavelength of 400nm induces 10S to gel.

Example 2

Hematopoietic stem cells (which are subsequently differentiated into osteoclasts) were placed in DMEM medium containing 4.8mmol/L of sugar, and hypoxic conditions of 1% O were maintained₂，5％CO₂，94％N₂Culturing for 6 days to make it more suitable for the microenvironment in hydrogel, and obtaining the pretreated hematopoietic stem cells.

Collecting plasma exosomes, dissolving in 5 × DMEM +150ng/ml PDGF +150ng/mLbFGF +5 × B27 culture medium, and performing electroporation treatment for 2 times under the conditions that the voltage is 600V, the capacitance is 100mF and the electrode spacing of an electric shocking cup is 4mm, so that the exosomes are rich in nutrition and key growth factors required by cell growth and survival, and the engineered exosomes are obtained.

And (3) performing pretreatment on the hematopoietic stem cells and engineered exosomes according to the number of the hematopoietic stem cells: exosome number was 1: 10⁵The components are mixed in a DMEM medium to obtain a mixed solution, and the mixed solution is mixed with 20% gelMA (Sovium Intelligent manufacturing research institute, GM-60) according to a volume ratio of 1:3, adding 0.3% (w/v) LAP photoinitiator, and inducing 10S to gel by blue light with the wavelength of 400 nm.

Example 3

Placing MSC in DMEM medium containing sugar 4.0mmol/L, and maintaining low oxygen condition 1% O₂，5％CO₂，94％N₂Culturing for 9 days to make it more suitable for the microenvironment in hydrogel to obtain pretreated hematopoietic stem cells.

Collecting plasma exosomes, dissolving in 9 × DMEM +250ng/ml PDGF +250ng/mLbFGF +9 × B27 culture medium, and performing electroporation treatment for 4 times under the conditions that the voltage is 800V, the capacitance is 200mF and the electrode spacing of an electric shocking cup is 4mm, so that the exosomes are rich in nutrition and key growth factors required by cell growth and survival, and the engineered exosomes are obtained.

And (3) performing pretreatment on the MSC and the engineered exosome according to the cell number of the MSC: exosome number was 1: 10⁵The components are mixed in a DMEM medium to obtain a mixed solution, and the mixed solution is mixed with 20% gelMA (Sovium Intelligent manufacturing research institute, GM-60) according to the volume ratio of 2:1 proportional isovolumetricMixing, adding 0.4% (w/v) LAP photoinitiator, and inducing 10S to gel by blue light with the wavelength of 400 nm.

Comparative example 1

The difference from example 1 is that it does not contain MSC and exosome, and the rest is the same as example 1, and is a cell-free group.

Comparative example 2

The difference from example 1 is that MSC was cultured in conventional DMEM medium without low-sugar hypoxia treatment and without exosome, and the cells were normally cultured in the same manner as example 1.

Comparative example 3

The difference from example 1 is that it does not contain exosomes, and the rest is the same as example 1. And is marked as an anoxic pretreatment cell group.

Comparative example 4

The difference from example 1 is that the exosomes were not treated by electroporation, and the remainder was the same as example 1, and they were a hypoxic pretreated cell + control exosome group.

The gel obtained in example 1 and comparative examples 1 to 4 was added to 1 × PBS, and after the gel was cultured in a cell incubator for 48 hours, the hydrogel was removed, frozen embedded, sliced, stained with Hoechst, and the cells were counted. The results are shown in FIG. 1.

As can be seen from FIG. 1, the cell survival rate can be effectively improved by carrying out hypoxia treatment and adding exosomes, the survival rate improving effect is better after the exosomes are subjected to engineering treatment, and the cell survival rate can be remarkably improved by the hydrogel provided by the invention.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.