A method for tissue regeneration combining foaming hydrogel and cell microspheres

Abstract

The present invention relates to a method for tissue regeneration combining foaming hydrogel and cell microspheres, and belongs to the field of biomedical technology. The method comprises preparing foaming hydrogel and cell microspheres, and three-dimensionally cultured adipose stem cell spheres are implanted in the foaming hydrogel. The present invention provides a tissue regeneration model constructed by a method for tissue regeneration combining foaming hydrogel and cell microspheres, and the application of the model; the present invention solves the key problems in tissue regeneration of high cell necrosis rate and poor stem cell differentiation in hydrogels; an effective solution for optimizing hydrogels is proposed, which promotes tissue regeneration to the maximum extent by integrating pore formation, cell compatibility, injectability and bioactivity, and optimizing combined seed cells.

Description

Technical Field

[0001] The invention relates to a method for tissue regeneration combining foaming hydrogel and cell microspheres, belonging to the technical field of biomedicine. Background Art

[0002] The construction of vascularized tissue-engineered fat provides a new option for repairing tissue (such as breast) defects. However, the lack of vascularized environment and adipose stem cells, as well as the low efficiency of adipogenic differentiation, hinder its application. Therefore, the technical field is in urgent need of a new culture method for constructing vascularized tissue-engineered fat, which should effectively promote cell survival, migration and adipogenic differentiation, and significantly promote the regeneration of vascularized adipose tissue in vivo. Provide a new solution for the regeneration of vascularized adipose tissue and establish a universal research model for breast repair and reconstruction. Summary of the invention

[0003] The purpose of the present invention is to solve the technical problem of how to effectively construct vascularized tissue engineering fat.

[0004] In order to achieve the purpose of solving the above problems, the technical solution adopted by the present invention is to provide a method for tissue regeneration combining foaming hydrogel and cell microspheres, including preparing foaming hydrogel and cell microspheres, and implanting three-dimensionally cultured adipose stem cell microspheres in the foaming hydrogel.

[0005] Preferably, the foaming hydrogel is foaming methacryloyl gelatin.

[0006] Preferably, the preparation of the cell microspheres comprises the following steps:

[0007] Step 1: Prepare a polydimethylsiloxane (PDMS) mold; use an aluminum alloy microporous plate as a template, the bottom of the aluminum alloy microporous plate includes a structural surface composed of square truncated cone protrusions; PDMS is mixed with a main agent A and a curing agent B in a ratio of 10:1, poured into a culture dish with an aluminum alloy microporous plate at the bottom, and degassed to remove bubbles; the obtained PDMS mold is separated from the template; a plurality of square truncated cone-shaped micropores are provided on the surface of the PDMS mold;

[0008] Step 2: Prepare an agarose microplate; soak the PDMS mold in 75% alcohol for 2 hours for sterilization, then remove it and rinse it with a sterile PBS solution; pour the agarose solution into the PDMS mold, remove the agarose after solidification, and place the agarose at the bottom of the 24-well plate well;

[0009] Step 3: In a 24-well plate containing agarose, add culture medium to each well and remove bubbles; then inoculate the cell suspension, with 3*10 6 The cells were cultured for 3 days to obtain adipose stem cell spheres.

[0010] The present invention provides a tissue regeneration model constructed according to the above-mentioned method for tissue regeneration combining foaming hydrogel and cell microspheres.

[0011] The present invention provides the application of the above tissue regeneration model in non-diagnostic methods and non-therapeutic methods.

[0012] The present invention provides regenerated tissue constructed according to the above-mentioned method for tissue regeneration combining foaming hydrogel and cell microspheres.

[0013] The present invention provides the use of the above-mentioned regenerative tissue in non-diagnostic methods and non-therapeutic methods.

[0014] Preferably, the application includes application in preparing tissue fillers.

[0015] Preferably, the application includes application in preparing breast tissue fillers.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention solves the key problems of high cell necrosis rate in hydrogel and poor stem cell differentiation in tissue regeneration. The foaming process does not require physical and chemical foaming agents and does not damage cells. The porous structure of the hydrogel promotes the diffusion of oxygen and nutrients, so that the surrounded cells have stronger survival ability. Endogenous cells and surrounding tissues are more easily infiltrated, resulting in the rapid formation of blood vessels in the hydrogel. The foaming process is fast and pollution-free, and does not require complex equipment, which is conducive to rapid promotion and clinical application. In the present invention, 3D adipose stem cell microspheres replace traditional monolayer cultured cells to achieve better differentiation and fat regeneration. The cells in the 3D adipose stem cell microspheres are in a state of micro-hypoxia, and the cells secrete more key angiogenic factors such as vascular endothelial growth factor (VEGF), which further promotes the formation of blood vessels in the graft. In addition, since the 3D adipose stem cell microspheres simulate the three-dimensional environment of cells in the body, the anti-apoptosis performance of the cells is also greatly improved, which is more conducive to cell survival.

[0018] The present invention proposes an effective solution for optimizing hydrogels, which maximizes tissue regeneration by integrating pore formation, cell compatibility, injectability and bioactivity, combined with optimization of seed cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the preparation process of foaming hydrogel.

[0020] Figure 2 Schematic diagram of the cell microsphere preparation process.

[0021] Figure 3 Schematic diagram of the preparation process of GelMA hydrogel and fat regeneration model.

[0022] Figure 4 This is a comparison of the surface vascularization of the stent two weeks after implantation in the body.

[0023] Figure 5 This is a diagram of the formation of aluminum alloy microplate and cell microspheres.

[0024] Figure 6 A comparison of the vascularization conditions in the four groups of hydrogels.

[0025] Figure 7 A comparison of adipogenesis in four groups of scaffolds. DETAILED DESCRIPTION

[0026] In order to make the present invention more clearly understood, a preferred embodiment is described in detail with reference to the accompanying drawings as follows:

[0027] like Figure 1-7 As shown, the present invention proposes a fat regeneration strategy that combines gas foaming hydrogel and three-dimensional stem cell microspheres. The hydrogel is optimized by using bubble foaming combined with photocuring technology to generate foamed methacryloyl gelatin (f-GelMA), which provides a new strategy for preparing non-toxic porous hydrogels and can significantly promote the vascularization of regenerated tissues. Three-dimensional cultured adipose stem cell spheres (s-ADSC) are used to replace two-dimensional cultured monolayer adipose stem cells (m-ADSC), and seed cells are optimized to promote adipogenic differentiation. The culture model constructed based on f-GelMA and s-ADSC can effectively promote cell survival, migration and adipogenic differentiation in vitro, and significantly promote the regeneration of vascularized adipose tissue in vivo. The present invention provides a solution for the regeneration of vascularized adipose tissue and establishes a universal research model for breast defect repair and reconstruction. In addition, the foaming technology proposed in the present invention can be applied to various hydrogels, and a variety of microenvironment regulators can be added in combination. Therefore, the system can also establish a universal research model for the regeneration of other tissues.

[0028] like Figure 1 Shown is a schematic diagram of the preparation process of the foaming hydrogel;

[0029] Hydrogel foaming: Gas foaming method has become a promising pore-making method because it avoids the use of low temperature and organic solvents and can prepare fluid hydrogels. In this regard, the present invention provides a uniformly foamable and injectable device, which uses the mechanical shear force generated during the pushing process to achieve uniform distribution of air in the hydrogel. This device cleverly uses a three-way tube 1, the two ends of the three-way tube 1 are connected to syringes respectively, and the third end is connected to a needle. The syringe at the first end is filled with hydrogel 2, and the syringe at the second end is filled with air 3. The syringes at the first and second ends are pushed several times for foaming, and then the cell ball 4 is added and pushed again in small amounts several times to mix the cell ball 4 and the hydrogel 2 evenly, and then injected into a specific mold through the needle end and irradiated with 405nm blue light to complete the curing of the f-GelMA hydrogel (the hydrogel and cell microspheres are mixed first and then injected into the mold through the needle end for curing). After injection, the cured hydrogel 2 completes the entire cell-carrying foaming process. The hydrogel is cured and formed by light irradiation.

[0030] like Figure 2 Shown is a schematic diagram of the cell microsphere preparation process;

[0031] Cell microsphere culture method:

[0032] 1. Preparation of polydimethylsiloxane (PDMS) mold:

[0033] A customized aluminum alloy microplate 5 (15.6 mm in diameter) was used as a template. The bottom of the microplate 5 contained approximately 300 square truncated cones 6 protrusions (e.g. Figure 5 Figure A in the middle shows a structural surface composed of a tetrahedral pyramid with an upper base of 270 μm, a lower base of 800 μm, and a height of 300 μm). PDMS (Dow Corning, USA) is prepared by mixing a main agent A and a curing agent B in a ratio of 10:1 (w / w). The mixture is poured into a small culture dish with a customized aluminum alloy microwell plate 5 at the bottom, and degassed to remove bubbles to obtain a PDMS mold 7. The PDMS mold 7 is placed in a 37°C incubator for 48 hours and carefully separated from the aluminum alloy microwell plate 5 template. There are approximately 300 concave square truncated pyramid-shaped microwells on the surface of the PDMS mold 7 (composed of an inverted tetrahedral pyramid with a lower base of 270 μm, an upper base of 800 μm, and a height of 300 μm)

[0034] 2. Preparation of agarose microplate:

[0035] Agarose 8 powder (2%, w / v) was dissolved in boiling water and sterilized in a glass bottle under high pressure steam. PDMS mold 7 was sterilized in 75% alcohol for 1 hour and washed 3 times in PBS. 0.5 mL of agarose solution was poured into the PDMS mold 7 containing microwells and placed at 4°C for 5 minutes. After solidification, the separated agarose microwell plate was taken out and placed at the bottom of a single well of the 24-well plate 9.

[0036] 3. Formation of s-ADSCs:

[0037] In a 24-well plate containing agarose, 500 μL of culture medium was added to each well and air bubbles were removed. Then the cell suspension was inoculated (3×10 cells per well). 6 cells), and gently blow several times to evenly distribute the cells in the wells. Then incubate the 24-well plate at 37°C, 5% CO2 for 3 days. After the adipose stem cell sphere s-ADSC is formed, the cell sphere is blown out with a pipette gun and then collected by centrifugation.

[0038] Example

[0039] like Figure 3 Shown is a schematic diagram of the preparation process of GelMA hydrogel and fat regeneration model.

[0040] Take GelMA hydrogel and fat regeneration model as an example:

[0041] ① Preparation stage: prepare two sterile syringes, a sterile three-way pipe, and a sterile needle. Add hydrogel and blue light initiator (LAP) to one syringe, and add air to the other syringe (the ratio of air to hydrogel will affect the pore size inside the foaming hydrogel). Connect the two syringes and a needle to the three-way pipe.

[0042] ② Foaming stage: Push the syringes at both ends to evenly distribute the air in the hydrogel. This process uses the shear force generated when the air passes through the small holes to achieve foaming. In practice, the foam produced by pushing 40 times has a high porosity and uniform pore size. Because excessive mechanical shear force may damage the cells, we add the cell spheroid suspension after the foaming is completed. Push again 10 times to mix the cell spheroids and hydrogel evenly. It has been confirmed by CCK-8 that pushing 10 times causes slight damage to the cells.

[0043] ③Injection and cross-linking stage: Open the injection switch of the three-way tube and inject the hydrogel into the lesion. After light cross-linking, the hydrogel completes the entire foaming hydrogel process.

[0044] like Figure 4 The figure shows the comparison of surface vascularization of the stent two weeks after implantation in the body. The figure A shows the non-foamed group, which shows poor vascularization; the figure B shows the foamed group of the present invention, which shows good vascularization.

[0045] Figure 5 Figure 1 shows the formation of aluminum alloy microplates and cell microspheres. Figure A shows the customized aluminum alloy microplate and the square truncated cone protrusions on the surface; Figure B shows the process of cell microsphere formation in a 24-well plate, after the first, second, and third days.

[0046] Figure 6The following are comparisons of the vascularization in four groups of hydrogels. Figure A shows the non-foamed monolayer cell group; Figure B shows the non-foamed cell sphere group; Group C shows the foamed monolayer cell group; and Group D shows the foamed cell sphere group. The results show that the foamed combined with microsphere group has the best vascularization.

[0047] Figure 7 The following is a comparison of the adipogenesis in the four groups of scaffolds. A is the peripheral group of non-foamed monolayer cells; B is the peripheral group of non-foamed cell spheres; C is the peripheral group of foamed monolayer cells; D is the peripheral group of foamed cell spheres; E is the internal group of non-foamed monolayer cells; F is the internal group of non-foamed cell spheres; G is the internal group of foamed monolayer cells; H is the internal group of foamed cell spheres. The results show that the adipogenesis of foamed combined microspheres is very good both inside and outside.

[0048] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and equivalent changes made by using the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for tissue regeneration combining foaming hydrogel and cell microspheres, characterized in that: The method comprises preparing foamed methacryloyl gelatin and cell microspheres, wherein the cell microspheres are three-dimensionally cultured fat stem cell spheres, and the cell microspheres are implanted in the foamed hydrogel. The method specifically comprises: A three-way tube is used, the two ends of the three-way tube are connected to syringes respectively, and the third end is connected to a needle. The syringe at the first end is filled with hydrogel, and the syringe at the second end is filled with air; the syringes at the first end and the second end are pushed 40 times for foaming, and then the cell microspheres are added and pushed again 10 times to mix the cell microspheres and the hydrogel evenly; then the f-GelMA hydrogel is injected into a specific mold through the needle end and irradiated with 405nm blue light to complete the curing of the hydrogel. After the injection, the hydrogel is cured to complete the entire cell-carrying foaming process, and the hydrogel is cured and formed by light irradiation; The preparation of the cell microspheres comprises the following steps: Step 1: Prepare a polydimethylsiloxane (PDMS) mold; use an aluminum alloy microporous plate as a template, the bottom of the microporous plate includes a structural surface composed of square truncated cone-shaped protrusions; PDMS is mixed with a main agent A and a curing agent B at a ratio of 10:1 w / w, poured into a culture dish with an aluminum alloy microporous plate at the bottom, and degassed to remove bubbles; the obtained PDMS mold is separated from the template; a plurality of square truncated cone-shaped micropores are provided on the surface of the PDMS mold; Step 2: Prepare an agarose microplate; pour the agarose solution into the PDMS mold, take out the agarose after solidification, and place the agarose at the bottom of the well of the 24-well plate; Step 3: In a 24-well plate containing agarose, add culture medium to each well and remove bubbles; then inoculate the cell suspension, with 3*10 6 The cells were cultured for 3 days to obtain adipose stem cell spheres.

2. A tissue regeneration model constructed by the method for tissue regeneration combining foaming hydrogel and cell microspheres according to claim 1.

3. The regenerated tissue constructed by the method for tissue regeneration combining foaming hydrogel and cell microspheres according to claim 1.

4. Use of the regenerated tissue according to claim 3 in preparing tissue fillers.

5. The use according to claim 4, characterized in that: The application includes application in preparing breast tissue fillers.

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