Catechol functionalized gelatin microspheres and application thereof in preparation of products for inhibiting formation of subcutaneous effusion and promoting tissue repair

Abstract

The invention provides a catechol functionalized gelatin microsphere and application of the catechol functionalized gelatin microsphere in preparation of a product for inhibiting formation of subcutaneous effusion and promoting tissue repair, and relates to the technical field of biological materials.According to the designed and prepared catechol functionalized gelatin microsphere, catechol groups are introduced into the gelatin microsphere, and the catechol functionalized gelatin microsphere is prepared; good liquid absorption performance and biodegradability are realized. Cell experiment results show that the catechol functionalized gelatin microspheres have super multiple interfaces and can promote cell adhesion and proliferation, and the loose structure of the catechol functionalized gelatin microspheres can promote cell migration. In-vivo rabbit chest wall defect repair experiment results show that the catechol functionalized gelatin microspheres not only can inhibit formation of subcutaneous effusion, but also can collect autologous cells in situ and promote proliferation of the autologous cells and secretion of extracellular matrixes (such as collagen). The results show that the catechol functionalized gelatin microsphere material has clinical application potentials of inhibiting formation of subcutaneous effusion and promoting healing of subcutaneous wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to catechol-functionalized gelatin microspheres and applications thereof in preparing products for inhibiting subcutaneous effusion formation and promoting tissue repair. Background Art

[0002] Breast cancer is the second most deadly cancer in women worldwide, accounting for 31% of female cancers. Resection is currently the usual treatment option. Subcutaneous effusion (seroma) is the most common complication after breast surgery. Due to the large area of ​​the free flap during breast cancer surgery, a large cavity is formed, and the surface skin and the basal muscles cannot be completely adhered together, resulting in subcutaneous effusion. The formation of subcutaneous effusion may cause more serious symptoms, especially for the elderly, because their ability to absorb effusion is poor. If subcutaneous effusion occurs repeatedly, it may increase the risk of infection, prolong hospitalization, delayed wound healing, flap necrosis, delayed adjuvant therapy, and even reoperation. So far, closed negative pressure suction drainage has become the standard of wound care.

[0003] In recent years, with the aging population and the increasing incidence of breast cancer, postoperative care after mastectomy has attracted more and more attention, among which preventing the formation of postoperative effusion has become a hot topic of current research. Negative pressure closed drainage is a commonly used clinical method, but its drainage tube is connected from the body wound to the outside of the body, increasing the risk of wound infection. In recent years, closed suction drainage, flap fixation, adhesion with adhesive tissue adhesive, breast reconstruction and other methods have been developed one after another. In addition, the combination of negative pressure drainage and suture reduces the formation of effusion and avoids the use of negative pressure drainage tubes. However, when the flap is fixed with sutures, it may cause skin depression and decreased shoulder function. Fibrin sealant is a biological hemostatic agent that affects the coagulation cascade reaction. Fibrin sealant helps to eliminate postoperative cavities, but the dense colloidal structure formed by it usually inhibits the migration of autologous cells to the defect, thereby hindering tissue regeneration. In addition, the use of prostheses such as silicone or autologous tissue for breast reconstruction will inhibit the formation of cavity effusion to a certain extent.

[0004] Tissue engineering aims to repair damaged tissues by using biomaterial scaffolds to load cells and / or bioactive molecules. It is recognized as the most promising means of tissue repair. Tissue engineering strategies have been studied for postoperative breast reconstruction, but there are few reports on the use of tissue engineering scaffold materials to inhibit postoperative effusion formation and subcutaneous wound healing after mastectomy.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide catechol functionalized gelatin microspheres (ca-CGMSs) and their use in the preparation of products for inhibiting the formation of subcutaneous effusion and promoting tissue repair, so as to at least solve one of the technical problems existing in the prior art.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The present invention provides a catechol functionalized gelatin microsphere, which is prepared by the following method:

[0009] (a) stirring and dispersing a mixed solution of gelatin and catechol-functionalized gelatin into an oil phase, cooling the mixture in an ice-water bath, and preparing physically cross-linked gelatin-based microspheres;

[0010] (b) chemically crosslinking the physically crosslinked gelatin-based microspheres obtained in step (a) using a chemical crosslinking agent to obtain the catechol-functionalized gelatin microspheres.

[0011] Furthermore, the catechol-functionalized gelatin includes gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid and / or gelatin-3,4-dihydroxybenzoic acid.

[0012] Furthermore, the concentration of the mixed solution of gelatin and catechol-functionalized gelatin is 8% to 30% (w / v).

[0013] Furthermore, the mass ratio of gelatin to catechol functionalized gelatin is 1:0.01 to 1:20.

[0014] Furthermore, the dry particle size of the catechol functionalized gelatin microspheres is 50-500 μm.

[0015] Furthermore, the solvent of the cross-linking system is a pure organic solvent or a water / organic solvent mixed system.

[0016] Further, the chemical cross-linking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N,N′-dicyclohexylcarbodiimide (DCC), genipin, formaldehyde and glutaraldehyde.

[0017] The present invention also provides the use of the catechol functionalized gelatin microspheres in preparing products for inhibiting the formation of subcutaneous effusion and / or promoting tissue repair.

[0018] Furthermore, the catechol-functionalized gelatin microspheres achieve tissue repair by promoting at least one of cell adhesion, proliferation and migration.

[0019] Furthermore, the catechol-functionalized gelatin microspheres achieve tissue repair by inhibiting the formation of effusion or absorbing effusion.

[0020] Furthermore, the tissue repair includes subcutaneous wound healing and tissue repair after breast cancer surgery.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Subcutaneous effusion (seroma) often forms after mastectomy, and poor absorption may lead to infection of subcutaneous tissue organs and even endanger life. The present invention designs and prepares catechol-functionalized gelatin microsphere materials, which have good liquid absorption performance and biodegradability. The results of cell experiments show that the ultra-large specific surface area of ​​catechol-functionalized gelatin microspheres can promote cell adhesion and proliferation, and its loose structure can also promote cell migration. The results of the in vivo rabbit chest wall defect repair experiment showed that catechol-functionalized gelatin microspheres not only have a good ability to inhibit the formation of subcutaneous effusion, but also can recruit cells in situ and promote their proliferation and secretion of extracellular matrix (such as collagen). These results show that catechol-functionalized gelatin microsphere materials have the function of inhibiting the formation of subcutaneous effusion and promoting subcutaneous tissue regeneration, and have clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The synthesis process and H-NMR characterization of gelatin-3,4-dihydroxyphenylacetic acid containing catechol groups provided in Example 1 of the present invention.

[0025] Figure 2 The synthesis process and H-NMR characterization of gelatin-dopamine containing catechol groups provided in Example 2 of the present invention.

[0026] Figure 3 The morphology and performance characterization results of ca-CGMSs provided in the experimental examples of the present invention; wherein, (A) ca-CGMSs powder; (B) scanning electron microscope (SEM) image of ca-CGMSs; (C) micrograph of swollen ca-CGMSs; (D) particle size distribution of ca-CGMSs; (E) swelling ratio of ca-CGMSs obtained under different preparation conditions; (F) degradation curve of ca-CGMSs.

[0027] Figure 4The results of viability and proliferation detection of L929 cells cultured on PFS and ca-CGMSs scaffolds provided in the experimental examples of the present invention are shown; wherein, (A) live / dead cell staining of L929 in PFS and ca-CGMSs scaffolds, respectively, the scale bar is 300 μm; (B) cell proliferation detection of L929 in PFS and ca-CGMSs scaffolds, respectively, **** indicates p < 0.0001.

[0028] Figure 5 This is a result diagram of the F-actin morphology detection of L929 cultured on PFS and ca-CGMSs scaffolds provided in the experimental examples of the present invention, with a scale bar of 300 μm.

[0029] Figure 6 The results of the migration test of L929 cells cultured on PFS and ca-CGMSs scaffolds provided in the experimental examples of the present invention are shown in Figure 1; (A) Crystal violet staining image of L929 cells that pass through PFS or ca-CGMSs scaffolds and enter the lower chamber of the transwell, with a scale bar of 300 μm; (B) Statistics of the number of migrated cells. Wherein, **** indicates p < 0.0001.

[0030] Figure 7 The experimental examples of the present invention provide a New Zealand rabbit mastectomy model construction and fluid effusion quantification result diagram; wherein, (A) the actual operation diagram of the New Zealand rabbit mastectomy model; (B) photos of the New Zealand rabbits during and after the operation; (C) statistics of subcutaneous fluid volume, wherein, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0031] Figure 8 The F-actin staining and nucleus DAPI staining signal statistics of the recruited cells after different scaffolds were implanted into the subcutaneous tissue defect for 7 and 28 days in the experimental examples of the present invention; wherein, (A) F-actin staining, scale bar is 300 μm; (B) cell nucleus quantitative statistics; wherein, **** indicates p < 0.0001.

[0032] Fig. 9 H&E and Masson staining of new tissue during the repair of subcutaneous tissue defects provided in the experimental example of the present invention, the scale bar is 300 μm.

[0033] Fig.10 This is the Sirius red staining of the new tissue during the repair of subcutaneous tissue defects provided in the experimental example of the present invention. The scale bar is 200 μm.

[0034] Fig.11 This is an experimental example of the present invention, providing immunostaining of type 1 collagen (Col1) in the new tissue during the repair of subcutaneous tissue defects. The scale bar is 300 μm.

[0035] Fig.12 This is a graph showing the comparison of the adhesion and subcutaneous tissue load-bearing capacity of gelatin microspheres without catechol groups and gelatin microspheres with catechol groups provided in the experimental examples of the present invention. DETAILED DESCRIPTION

[0036] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0037] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0038] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The inventors of the present invention have considered the huge clinical application potential of cell-free scaffold materials, and tissue-inductive scaffold materials have become a hot spot in tissue engineering research. Gelatin is a natural polymer derived from the denaturation of animal structural collagen, which has good biocompatibility, biodegradability, non-toxicity and low immunogenicity. Gelatin microspheres are often compounded into traditional three-dimensional scaffolds and used as cell or drug carriers. Experiments have found that catechol can mediate the formation of a series of non-covalent bonds between the material and the surface of biological tissues, promoting their adhesion; and the catechol group has antibacterial, anti-inflammatory and antioxidant functions. However, catechol-functionalized gelatin microspheres and their use in inhibiting the formation of subcutaneous effusions and promoting subcutaneous tissue repair have not been reported.

[0040] Based on this, the first aspect of the present invention provides a catechol functionalized gelatin microsphere, which is prepared by the following method:

[0041] (a) stirring and dispersing a mixed solution of gelatin and catechol-functionalized gelatin into an oil phase, cooling the mixture in an ice-water bath, and preparing physically cross-linked gelatin-based microspheres;

[0042] (b) chemically crosslinking the physically crosslinked gelatin-based microspheres obtained in step (a) using a chemical crosslinking agent to obtain the catechol-functionalized gelatin microspheres.

[0043] The present invention obtains catechol functionalized gelatin microsphere material by introducing catechol groups, which has good liquid absorption performance and biodegradability. The results of cell experiments show that the super large specific surface area of ​​catechol functionalized gelatin microspheres can promote cell adhesion and proliferation, and its loose structure can also promote cell migration. The results of the in vivo rabbit chest wall defect repair experiment showed that catechol functionalized gelatin microspheres not only have a good ability to inhibit the formation of subcutaneous effusion, but also can recruit cells in situ and promote their proliferation and secretion of extracellular matrix (such as collagen). These results show that catechol functionalized gelatin microsphere material has the function of inhibiting the formation of subcutaneous effusion and promoting the regeneration of subcutaneous tissue, and has clinical application potential.

[0044] In some preferred embodiments, the catechol-functionalized gelatin includes gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid and / or gelatin-3,4-dihydroxybenzoic acid; gelatin-dopamine alone, gelatin-3,4-dihydroxyphenylacetic acid alone, gelatin-3,4-dihydroxybenzoic acid alone can be selected and mixed with gelatin, or any two of gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid and gelatin-3,4-dihydroxybenzoic acid or a mixture of all three can be selected and mixed with gelatin-dopamine; when a mixture is selected, the ratio of gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid and gelatin-3,4-dihydroxybenzoic acid is not limited.

[0045] In some preferred embodiments, the concentration of the mixed solution of gelatin and catechol-functionalized gelatin is 8% to 30% (w / v), for example, but not limited to 8%, 10%, 15%, 20%, 25% or 30%.

[0046] In some preferred embodiments, the ratio of gelatin to catechol functionalized gelatin is 1:0.01 to 1:20, for example, it can be, but not limited to, 1:0.01, 1:1, 1:5, 1:10, 1:15 or 1:20. When the ratio of gelatin to catechol functionalized gelatin is within the above ratio range, increasing the amount of catechol functionalized gelatin can promote the water absorption capacity and tissue adhesion performance of the obtained microspheres. When the amount of catechol functionalized gelatin is too high, the cross-linking density of the catechol functionalized gelatin microspheres will be reduced, accelerating degradation.

[0047] In some preferred embodiments, the dry particle size of the catechol functionalized gelatin microspheres is 50-500 μm, for example, but not limited to 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm or 500 μm. When the dry particle size of the catechol functionalized gelatin microspheres is within the above ratio range, the obtained microspheres can not only absorb tissue effusion and adhere to the tissue surface, but also quickly recruit cells and promote cell proliferation.

[0048] In some preferred embodiments, the solvent of the cross-linking system is a pure organic solvent or a water / organic solvent mixed system. For example, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, methanol, acetone, etc. can be used as pure organic solvents, or 40% ethanol aqueous solution, 60% dimethyl sulfoxide aqueous solution, 90% acetone aqueous solution, etc. can be used as a water / organic solvent mixed system.

[0049] According to the characteristics of the catechol functionalized gelatin microspheres provided by the present invention, the present invention also provides the use of the catechol functionalized gelatin microspheres in preparing products for inhibiting the formation of subcutaneous effusion and / or promoting tissue repair.

[0050] In some optional embodiments, the catechol-functionalized gelatin microspheres achieve tissue repair by promoting at least one of cell adhesion, proliferation and migration.

[0051] Furthermore, the catechol-functionalized gelatin microspheres achieve tissue repair by inhibiting the formation of effusion or absorbing effusion.

[0052] Subcutaneous effusion (seroma) often forms after mastectomy, and poor absorption may lead to infection of subcutaneous tissues and organs, and even endanger life. The catechol-functionalized gelatin microspheres provided by the present invention can effectively promote subcutaneous wound healing and tissue repair after breast cancer surgery.

[0053] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0054] Example 1

[0055] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, comprising the following steps:

[0056] 1. Synthesis of Catechol-functionalized Gelatin Macromer

[0057] Synthesis of 3,4-dihydroxyphenylacetic acid modified gelatin macromer: Dissolve 3,4-dihydroxyphenylacetic acid (2 g) in 200 ml PBS (pH = 7) in a 40°C water bath, add EDC (2.0 g) and NHS (1.4 g), adjust the solution pH to 4-6, stir for 30 minutes and then add gelatin (2 g), react for 36 hours and precipitate into industrial ethanol, dialyze in pure water for 3 days and then freeze-dry to obtain gelatin-3,4-dihydroxyphenylacetic acid. H NMR spectrum ( 1 H NMR) characterization showed the successful synthesis of gelatin-3,4-dihydroxyphenylacetic acid rich in catechol groups ( Figure 1 ).

[0058] 2. Preparation of Physically Cross-linked Gelatin Microspheres

[0059] Gelatin microspheres were prepared using the oil-in-water method. Specifically, 1.5 g of gelatin and 1.5 g of catechol-functionalized gelatin were added to 20 ml of double distilled water and dissolved in an oven at 40 °C. The gelatin solution was then poured into 80 ml of mixed oil (paraffin oil: edible oil = 3:1) preheated at 45 °C and stirred on a magnetic stirrer at 45 °C for 30 min. The reaction system was then transferred to an ice / water bath (0 °C) and stirred for another 30 min. The precipitate was then washed 3 times with 1,4-dioxane and acetone, and then washed twice with anhydrous ethanol to obtain physically cross-linked gelatin microspheres.

[0060] 3. Preparation of ca-CGMSs

[0061] The physically cross-linked gelatin microspheres were placed in 30 mL of PBS / ethanol (3:7 v / v), EDC (0.6 g) and N-hydroxysuccinimide (NHS, 0.42 g) were added, and the microspheres were placed horizontally and shaken for a certain period of time to obtain chemically cross-linked gelatin microspheres (ca-CGMSs), which were washed three times with double distilled water and dried to obtain a light yellow powder ( Figure 3 A) in the sample was stored in a 4°C refrigerator for later use.

[0062] Example 2

[0063] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, which is different from Embodiment 1 in that:

[0064] 1. Synthesis of Catechol-functionalized Gelatin Macromer

[0065] Synthesis of dopamine-modified gelatin macromer: Dissolve gelatin (3 g) in 200 ml PBS (pH = 7) in a 40°C water bath, add EDC (1.5 g) and NHS (1 g), adjust the solution pH to 4-6, stir for 30 minutes, add dopamine hydrochloride (1.5 g), react for 36 hours, precipitate into industrial ethanol, dialyze in pure water for 3 days, and then freeze-dry to obtain gelatin-dopamine. H NMR spectrum ( 1 HNMR) characterization showed the successful synthesis of gelatin-dopamine rich in catechol groups ( Figure 2 ).

[0066] Example 3

[0067] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, which is different from Embodiment 1 in that:

[0068] In step 2, 0.3 g gelatin and 5.7 g catechol-functionalized gelatin were added to 20 ml double distilled water.

[0069] Example 4

[0070] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, which is different from Embodiment 1 in that:

[0071] In step 2, 0.8 g gelatin and 0.8 g catechol-functionalized gelatin were added to 20 ml double distilled water.

[0072] Example 5

[0073] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, which is different from Embodiment 1 in that:

[0074] In step 3, use DCC instead of EDC.

[0075] Example 6

[0076] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, which is different from Embodiment 1 in that:

[0077] In step 3, genipin was used instead of EDC.

[0078] Example 7

[0079] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, which is different from Embodiment 1 in that:

[0080] In step 2, 1 g of gelatin and 26 g of catechol-functionalized gelatin were added to 90 ml of double distilled water.

[0081] Example 8

[0082] This embodiment provides a method for preparing catechol functionalized gelatin microspheres, which is different from Embodiment 1 in that:

[0083] In step 2, 1.5 g gelatin and 1.5 g catechol-functionalized gelatin were added to 30 ml double distilled water.

[0084] Comparative Example 1

[0085] This comparative example provides a method for preparing gelatin microspheres, which differs from Example 2 in that:

[0086] In step 2, 3 g of gelatin was added to 20 ml of double distilled water to prepare catechol-free gelatin microspheres.

[0087] In order to save the experimental cost, the following experimental example is verified by taking the catechol functionalized gelatin microspheres provided in Example 2 as an example.

[0088] Experimental Example 1 Characterization of ca-CGMSs

[0089] SEM: The morphology of ca-CGMSs after gold spraying was observed by scanning electron microscopy.

[0090] The dried ca-CGMSs were sprayed with gold and then observed under a scanning electron microscope. It was found that the ca-CGMSs used in Example 2 were round particles with relatively smooth surfaces ( Figure 3 B).

[0091] Swelling rate: The dried ca-CGMSs were immersed in PBS and their morphology was observed after 48 hours. Similar to the SEM observation, they showed a relatively regular spherical shape ( Figure 3 C), the particle size distribution is 30 to 350 microns ( Figure 3 D in the figure). The surface solution was then carefully removed and weighed. The swelling ratio was calculated by comparing the weight at the time of drying. It was found that the cross-linking time of ca-CGMSs had little effect on its swelling ratio ( Figure 3 Therefore, Example 2 selected the shorter 6-hour crosslinking method. ca-CGMSs can absorb 7 times its own weight of PBS ( Figure 3 E) in the figure shows strong liquid absorption capacity.

[0092] In vitro enzymatic hydrolysis: ca-CGMSs (~150 mg) fully swollen in water were placed in 0.001% pancreatic enzyme solution (1 ml) at 37°C, and the surface solution was carefully removed at the predetermined time point before weighing. Compared with the initial weight, the degradation rate at different times was calculated.

[0093] The results showed that the fully swollen ca-CGMSs showed good degradation in 0.001% (w / v) pancreatic enzyme solution at 37°, and the degradation rate was almost proportional to the degradation time. The ca-CGMSs could be completely degraded in about 5 hours. The biodegradability of ca-CGMSs laid a good foundation for its use as a scaffold material for tissue repair ( Figure 3 F).

[0094] Experimental Example 2 Cell Experiment

[0095] 1. Cell Culture

[0096] 2×10 6 L929 mouse fibroblasts were inoculated into 10-cm culture dishes and 1640 complete medium (containing 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / ml streptomycin) was added. The culture dishes were marked and placed in a 37°C CO2 (5%) cell culture incubator. When the cells were in the logarithmic growth phase, they were digested with trypsin and counted.

[0097] 2. Cell Seeding

[0098] Prepare 1% agarose solution, add 200 μl of hot agarose solution to each well of a 24-well plate, and solidify in a refrigerator at 4 degrees. Resuspend 1 g of sterile ca-CGMSs in 1 ml of cell culture medium and inoculate into a 24-well plate, with 60 μl of ca-CGMSs suspension per well. In addition, add 100 μl of PFS to each well of the 24-well plate according to the instructions and solidify as a reference group. Add 2.5×10 5 cells and 1 ml of complete medium.

[0099] 3. Dead / Live Cell Staining

[0100] The material / cell complex was transferred to a 24-well plate and washed three times with sterile basal culture medium. Then, 1 ml of the prepared staining solution (1640 basal culture medium containing 2 μM calcein-AM and 4 μM propidium iodide-PI) was added to each well and incubated at 37°C in the dark for 30 min. After soaking twice in 1640 basal culture medium, the plates were observed under a fluorescence microscope.

[0101] In the test results, live cells and dead cells showed green and red fluorescence respectively ( Figure 4A in the figure). One day after cell inoculation, staining observation revealed that L929 fibroblasts had adhered to the gelatin microspheres and exhibited a normal polygonal shape, and the number of dead cells was relatively small. In the PFS group, most of the cells had adhered and stretched, and also exhibited a normal morphology, but the number of dead cells was significantly more than that in the ca-CGMSs group. These observations indicate that ca-CGMSs can provide a suitable microenvironment for the survival, adhesion and stretching of L929 fibroblasts. On the third day of culture, this example found that compared with the first day, the density of live cells on CA-CGMSS increased significantly, and the number of dead cells was relatively small. It was also found that the density of live cells on PFS also increased, but the number of dead cells was still more than that in the CA-CGMSS group. Similarly, compared with the third day, the cell density on ca-CGMSs and PFS increased again after 5 days of culture, and the number of dead cells in the PFS group was significantly more than that in the ca-CGMSs group. These results indicate that compared with PFS, ca-CGMSs can not only provide a better microenvironment for the survival of L929 fibroblasts and promote cell adhesion and extension, but also its unique three-dimensional structure and larger specific surface area can provide more growth space for cell proliferation and promote cell proliferation.

[0102] 4. Cell Proliferation Assay

[0103] The viability of L929 cells in the material / cell complex was detected using the PrestoBlue kit. According to the instructions, the PrestoBlue working solution was prepared. At the predetermined time point, the cell culture medium was replaced with 1 ml of the PrestoBlue working solution and incubated at 37°C in the dark for 30 minutes. The fluorescence was quantitatively analyzed at 560 / 590 nm (excitation / emission) using an ELISA reader.

[0104] The quantitative fluorescence intensity of the PFS group cells on the first day was used as a reference (set to 1) to obtain the relative cell numbers of each group at different time points ( Figure 4 B). In the ca-CGMS and PFS groups, the number of cells showed an increasing trend from day 1 to day 3 and day 5, a result consistent with the increase in cell density observed in the live / dead cell staining experiment results. At the same time, this example found that from day 1 to day 3 (PFS group: 6 times; ca-CGMSs group: 5.7 times), the cell proliferation rate on the two materials was significantly higher than the cell proliferation rate from day 3 to day 5 (PFS group: 1.6 times; ca-CGMSs group: 1.7 times). In addition, this example observed that on days 3 and 5, the number of cells in the ca-CGMSs group was significantly greater than that in the PFS group. These findings once again indicate that ca-CGMSs can provide a good cell growth microenvironment for the proliferation of L929 fibroblasts.

[0105] 5. Phalloidin Staining

[0106] In order to investigate the adhesion of cells on the material, the material / cell complex was stained with phalloidin in this embodiment. At a predetermined time, the material / cell complex was transferred to a 1.5 ml centrifuge tube, fixed with 4% paraformaldehyde for 30 minutes, then washed 3 times with PBS, and PBS containing 0.5% Triton X-100 and 3% BSA was added to permeabilize the cells for 15 minutes, and washed 3 times with PBS. After adding the phalloidin working solution and incubating in the dark for 30 minutes, it was counterstained with DAPI for 30 seconds, washed 3 times with PBS, and the cell distribution and cell morphology were observed under a fluorescence microscope.

[0107] Experimental findings ( Figure 5 ), L929 cells can express cytoskeleton microfilaments F-actin on both ca-CGMSs and PFS, promoting their adhesion and extension; cells in both groups appear flat polygonal. Together with nuclear staining, this example found that as the culture time increases, the cells in the PFS group gradually become crowded, which is not conducive to further cell proliferation; the cell density in the ca-CGMSs group also gradually increases, but it still has more cell growth space. These observations further indicate that the ca-CGMSs scaffold can provide more growth space for cell adhesion and proliferation. Careful observation will reveal that after 3 days of culture, some microspheres have been "bonded" together by cells, and as the culture reaches the 5th day, ca-CGMSs and cells form a three-dimensional whole, and the cells maintain normal polygons. However, cells in the PFS group can only grow on the surface of PFS. When cultured to the 5th day, this example found that the cells began to round, indicating that they may slowly die. It can be seen that compared with the PFS scaffold, ca-CGMSs can provide a better microenvironment for the adhesion, extension and proliferation of L929 cells.

[0108] 6. Cell Migration Assay

[0109] Transwell chambers were used to investigate the migration of cells in ca-CGMSs scaffolds. The Transwell chambers were placed in 24-well plates, and ca-CGMSs or PFS were added. After PFS solidified, L929 cell suspensions (5×10 4 cells / 200 μL), then 600 μL of complete culture medium was added to the lower chamber, which was placed in a 37°C cell culture incubator containing 5% CO2 and cultured for 24 h. The upper chamber was taken out, the cells inside the chamber were gently wiped off with a cotton swab, and the cells were stained with 0.1% crystal violet and observed under an optical microscope.

[0110] like Figure 6As shown, after 24 h, a large number of cells in the ca-CGMSs group migrated through the ca-CGMSs scaffold and invaded to the lower chamber of the chamber, while no cell migration to the lower chamber was observed in the PFS group, indicating that ca-CGMSs can better promote the migration of L929 cells compared with PFS.

[0111] Experimental Example 3 In vivo experiment

[0112] 1. Establishment and repair of mastectomy model

[0113] Female New Zealand rabbits weighing about 2 kg were selected. All animal experimental operations followed the regulations of the Ethics Committee of the Affiliated Hospital of Qingdao University. The experimental animals were randomly divided into a blank control group (n=6), a PFS group (n=6) and a gelatin microsphere (ca-CGMSs) group (n=6). Anesthesia was completed by intraperitoneal injection of sodium pentobarbital (3%, 40 mg / kg). The rabbits were placed in a supine position, depilated, and disinfected with povidone-iodine. A midline incision was made from the jugular notch to the xiphoid process with a scalpel and surgical scissors. The right chest skin flap was lifted and separated from the muscle tissue with hemostats to expose the pectoralis major, axilla, clavicle and latissimus dorsi edges. The pectoralis major, pectoralis minor, subcutaneous fat, mammary gland and visible lymph nodes were cut off. The defect size was 5 cm×3 cm, and a mastectomy model ( Figure 7 A and B in the figure). The defects were filled with PFS or ca-CGMSs, respectively marked as PFS group and ca-CGMSs group; in addition, the blank group did not receive any filling treatment. A drainage tube was placed in each group, led out subcutaneously, and connected to an external negative pressure ball to collect the drainage fluid. The wounds were closed with interrupted sutures, and the animals were monitored daily for wound dehiscence and infection, and the drainage fluid was weighed. At 7 and 28 days after surgery, the New Zealand rabbits were euthanized, and the defects and surrounding tissues were collected for subsequent histological analysis.

[0114] The experiment found that the amount of subcutaneous fluid in all groups after surgery gradually decreased with time. On the 1st, 2nd, and 3rd days after surgery, the amount of fluid collected in the blank control group was significantly more than that in the ca-CGMSs group and the PFS group ( Figure 7 C), where on day 1, the amount of subcutaneous fluid in the blank group was 3.6 times that of the ca-CGMSs group and 2.5 times that of the PFS group. By day 5, the volume of fluid collected in all groups was significantly reduced, and the amount of fluid in the ca-CGMSs group was significantly lower than that in the PFS group. Interestingly, at each time point, the average amount of fluid in the ca-CGMSs group was always less than that in the blank control group and the PFS group. In addition, the total amount of fluid collected in the ca-CGMSs group for 5 days was significantly less than that in the blank control group and the PFS group. In summary, a large amount of fluid will form in the subcutaneous cavity, and compared with PFS, ca-CGMSs shows better inhibition of subcutaneous fluid formation or absorption of fluid.

[0115] 2. Histomorphological staining

[0116] At 7 and 28 days after surgery, the present embodiment collected tissue samples from the tissue defect and the entire thickness of the surrounding tissue, and the tissue specimens were immersed in a 4% paraformaldehyde solution for 48 hours, and then dehydrated, paraffin-embedded, and cut into 5-micron-thick slices using a paraffin slicer and attached to an anti-shedding slide. After the tissue slices were hydrated, hematoxylin-eosin (H&E) staining, Masson trichrome staining, and Sirius red staining were performed. F-actin staining was performed using rhodamine-labeled phalloidin to examine cell distribution. For immunofluorescence staining of type I collagen, the hydrated sections were incubated in a PBS solution containing 3% BSA (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich) for 30 min to prevent nonspecific adsorption, then incubated in a type I collagen primary antibody solution at room temperature for 60 min, washed twice with PBS (15 min each time), incubated in 5 μg / ml IgG secondary antibody for 60 min, then incubated in 1 μg / ml DAPI solution for 5 min to counterstain the cell nuclei, immersed in PBS twice (15 min each time), and observed under a fluorescence microscope (Ti2, NiKon).

[0117] like Figure 8 As shown, at each time point, the F-actin and cell (nucleus) density of the ca-CGMSs group at the tissue defect was significantly higher than that of the blank control and PFS groups. Seven days after surgery, the cell (nucleus) density of the repair area in the blank group was significantly lower than that of the PFS group and the ca-CGMSs group. The number of cells was from most to least in the ca-CGMSs group, the PFS group and the blank group, and the trend of F-actin was consistent with the trend of the cell nucleus; and only in the ca-CGMSs group was the cell arrangement tropism. By the 28th day, the corresponding F-actin and cell (nucleus) densities of the ca-CGMSs group and the PFS group were significantly higher than those on the 7th day, indicating that more cells migrated to the defect site. However, the F-actin and cell (nucleus) densities of the ca-CGMSs group were still significantly higher than those of the blank control and PFS groups. In addition, this example found that F-actin and cells (nuclei) in the PFS group were mostly distributed at the edge of the defect, and fewer cells were distributed inside. However, in the ca-CGMSs group, not only was the density of F-actin and cells (nuclei) higher, but the cells were almost evenly distributed throughout the defect, and the cell arrangement was still directional. These results indicate that ca-CGMSs can recruit more cells to migrate to the muscle defect site and show a directional distribution, which will be beneficial to tissue defect repair.

[0118] In this example, H&E and Masson staining were performed on each group ( Fig. 9). By observing the H&E staining pictures, this embodiment found that there were significant differences in the repair of the defect sites in the three groups 7 days after surgery. The extracellular matrix (ECM) of the blank group was scattered, and the ECM density was significantly less than that of the PFS group and the ca-CGMSs group. The coloring of the middle part of the PFS group may be partly derived from the PFS itself, and the ECM arrangement of the PFS group was also scattered and disordered. The difference is that the ECM of the ca-CGMSs group is denser and more directional. Similarly, 28 days after surgery, the ECM in the defect repair area of ​​the ca-CGMSs group was denser than that of the PFS group and the blank control group. It can be seen that ca-CGMSs can provide a more ideal microenvironment for cells to migrate to the defect site, adhere to and grow, and promote their secretion of ECM. However, although PFS can adhere to the skin and subcutaneous tissue to a certain extent and inhibit the formation of effusion, its dense gel structure limits the migration of cells to the defect site and also hinders the formation of new tissue. Through careful observation, this example found that by the 28th day, the density of the ECM formed by the repair of the defect in the ca-CGMSs group was not much different from that of the undamaged skin tissue, suggesting that dense connective tissue has formed at the defect site. In order to further explore the type of collagen components in the new ECM, this example performed Masson staining on it, and the ECM distribution was similar to the results of HE staining observations: at each time point, the ECM density of the ca-CGMSs group was significantly higher than that of the PFS group and the blank group. The ECM coloring is mainly blue, with a small amount of red, indicating that the new ECM is mainly collagen fibers, with a small amount of muscle fibers, further indicating that the new tissue is mainly connective tissue, not muscle tissue.

[0119] In order to distinguish the types of collagen fibers, the tissues were stained with Sirius red and observed under a polarized light microscope. Fig.10 As shown in the figure, collagen fibers are mostly orange-yellow, indicating that the collagen component of the regenerated tissue is mainly type I collagen fibers. At two time points, the number of type I collagen fibers in the blank group was significantly less than that in the PFS group, and they were arranged in a scattered and disordered manner. The PFS group had fewer type I collagen fibers than the ca-CGMSs group. The collagen fibers in the ca-CGMSs group were arranged more tightly than those in the other two groups, and on the 28th day, the collagen fibers had filled the repair area.

[0120] Finally, this example used immunohistochemistry to stain type 1 collagen (Col1), a major ECM component. Fig.11As shown, similar to the results of histochemical staining, 7 days after surgery, the number of cells in the defect site of the blank group was small, and the Col1A1 secreted was lower than that of the other two groups. The ca-CGMSs group had the largest number of cells, and the amount of Col1A1 secreted was significantly higher than that of the other two groups. 28 days after surgery, compared with 7 days after surgery, the number of cells in the three groups increased, but the density of Col1A1 in the ca-CGMSs group was the highest, and it basically covered the defect site. These results show that the ca-CGMSs scaffold material can not only promote cell proliferation, but also promote the secretion of Col1A1, thereby promoting the in situ repair of subcutaneous tissue defects.

[0121] As a reference group, the present invention prepared gelatin microspheres without catechol groups according to Comparative Example 1 and implanted them into the subcutaneous defect. Fig.12 As shown, the catechol-containing gelatin microspheres can adhere well together and can bear the weight of the subcutaneous tissue ( Fig.12 A in the figure), however, although the gelatin microspheres without catechol groups were aggregated together, they were not tightly bonded to each other and could not bear the weight of the subcutaneous tissue ( Fig.12 B).

[0122] It should be noted that all quantitative data in the examples of the present invention were tested no less than three times, and the data are presented as mean ± standard deviation (SD). The data between the two groups were compared using a t-test. p < 0.05 between the two groups of data was considered statistically significant.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A catechol functionalized gelatin microsphere, characterized in that: Prepared by the following method: (a) stirring and dispersing a mixed solution of gelatin and catechol-functionalized gelatin into an oil phase, cooling the mixture in an ice-water bath, and preparing physically cross-linked gelatin-based microspheres; (b) chemically crosslinking the physically crosslinked gelatin-based microspheres obtained in step (a) using a chemical crosslinking agent to obtain the catechol-functionalized gelatin microspheres.

2. The catechol functionalized gelatin microspheres according to claim 1, characterized in that: The catechol functionalized gelatin includes gelatin-dopamine, gelatin-3,4-dihydroxyphenylacetic acid and / or gelatin-3,4-dihydroxybenzoic acid.

3. The catechol functionalized gelatin microspheres according to claim 1, characterized in that: The concentration of the mixed solution of gelatin and catechol functionalized gelatin is 8% to 30% (w / v).

4. The catechol functionalized gelatin microspheres according to claim 1, characterized in that: The mass ratio of gelatin to catechol functionalized gelatin is 1:0.01 to 1:

20.

5. The catechol functionalized gelatin microspheres according to claim 1, characterized in that: The dry particle size of the catechol functionalized gelatin microspheres is 50 to 500 μm.

6. The catechol functionalized gelatin microspheres according to claim 1, characterized in that: The solvent of the cross-linking system is a pure organic solvent or a water / organic solvent mixed system.

7. The catechol functionalized gelatin microspheres according to claim 1, characterized in that: The chemical cross-linking agent includes at least one of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, N,N′-dicyclohexylcarbodiimide, genipin, formaldehyde and glutaraldehyde.

8. Use of the catechol functionalized gelatin microspheres according to any one of claims 1 to 7 in the preparation of a product for inhibiting the formation of subcutaneous effusion and / or promoting tissue repair.

9. The use according to claim 8, characterized in that: The catechol-functionalized gelatin microspheres achieve tissue repair by promoting at least one of cell adhesion, proliferation and migration; Preferably, the catechol-functionalized gelatin microspheres promote tissue repair by inhibiting the formation of effusion or absorbing effusion.

10. The use according to claim 8 or 9, characterized in that: The tissue repair includes wound healing and tissue regeneration after breast cancer surgery.

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