Application of gradient degradation medical sponge as cell reservoir and / or carrier

Abstract

The invention discloses application of gradient degradation medical sponge as a cell reservoir and / or carrier. The preparation method of the medical sponge comprises the following steps: constructing a rigid part: cross-linking a first water-soluble cellulose derivative to form hydrogel slurry with first viscosity; constructing a flexible part: cross-linking a second water-soluble cellulose derivative to form hydrogel slurry with second viscosity; the hydrogel slurry with the first viscosity and the hydrogel slurry with the second viscosity are mixed to form mixed slurry, and the mixed slurry is subjected to vacuum freeze drying treatment to form the sponge. The sponge is capable of absorbing and containing cells such as MSC, chondrocytes or early osteoblasts, thereby serving as a reservoir and / or carrier for cells.

Description

Technical Field

[0001] The present invention relates to a new use of a medical sponge with gradient degradation. Specifically, it relates to the application of a medical sponge with gradient degradation as a cell reservoir and / or carrier. Background Art

[0002] In the prior art, medical sponges are mainly used for hemostasis. For example, Chinese Patent CN113599565B discloses a medical sponge with gradient degradation, which comprises a porous composite structure composed of a rigid part and a flexible part. Among them, the rigid part comprises a crosslinked first water-soluble cellulose derivative, and the flexible part comprises a crosslinked second water-soluble cellulose derivative; the porous composite structure swells when contacting an aqueous solution to form a hydrogel with elastic support, and, the flexible part degrades by hydration prior to the rigid part, forming gradient degradation.

[0003] This medical sponge with gradient degradation is extremely rich in elastic support, extremely elastic after absorbing water, has strong supporting force, can fully compress and stop bleeding at the wound surface, and can achieve an instantaneous hemostasis function; in addition, this medical sponge can achieve gradient degradation regulation. The crosslinking bonds of the molecules in its rigid structure are stronger than those in the flexible structure. The part composed of the flexible structure in the sponge structure degrades within 12 - 72 hours to form loose pores. When used in the nasal cavity, it can gradually restore the nasal ventilation function and improve the treatment comfort of patients; the rigid structure part degrades within 3 - 28 days, which can effectively isolate the wound surface and avoid wound adhesion.

[0004] Therefore, the main use of the medical sponge with gradient degradation in the prior art is instantaneous hemostasis, and it can achieve gradient degradation regulation. So far, there has been no report on the use of this medical sponge as a cell reservoir and / or carrier for absorbing and accommodating cells, especially for absorbing and accommodating human bone marrow mesenchymal stem cells (MSCs), chondrocytes or early osteoblasts. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of a medical sponge with gradient degradation as a cell reservoir and / or carrier. The preparation of the medical sponge with gradient degradation comprises the following steps:

[0006] Constructing the rigid part: providing a first water-soluble cellulose derivative, crosslinking it to form a hydrogel slurry with a first viscosity, the first viscosity being 110000 - 300000 mPa·s, the viscosity test concentration being 2%, and the temperature being 20°C;

[0007] Constructing the flexible part: providing a second water-soluble cellulose derivative, crosslinking it to form a hydrogel slurry with a second viscosity, the second viscosity being 20000 - 100000 mPa·s, the viscosity test concentration being 2%, and the temperature being 20°C;

[0008] Construct a composite structure: Mix a hydrogel slurry with a first viscosity and a hydrogel slurry with a second viscosity to form a mixed slurry;

[0009] Perform vacuum freeze-drying treatment on the mixed slurry to form a sponge.

[0010] In some embodiments of the present invention, the cells include MSCs, chondrocytes, or early osteoblasts.

[0011] In some embodiments of the present invention, the first water-soluble cellulose derivative forms a hydrogel slurry with a first viscosity through cross-linking. The first viscosity is 110,000 - 300,000 mPa·s, or 120,000 - 210,000 mPa·s, or 180,000 - 300,000 mPa·s. The viscosity test concentration is 2%, and the temperature is 20°C; the second water-soluble cellulose derivative forms a hydrogel slurry with a second viscosity through cross-linking. The second viscosity is 20,000 - 100,000 mPa·s, or 50,000 - 80,000 mPa·s, or 20,000 - 70,000 mPa·s. The viscosity test concentration is 2%, and the temperature is 20°C.

[0012] In some embodiments of the present invention, the cross-linking agents used in the cross-linking reactions of the first water-soluble cellulose derivative and the second water-soluble cellulose derivative are independently selected from one or more of formaldehyde, glyoxal, glutaraldehyde, carbodiimide, epichlorohydrin, tetramethylethylenediamine, phenylmaleimide, and N,N-methylenebisacrylamide. Those skilled in the art can determine appropriate cross-linking reaction conditions according to the gradient degradation requirements of the present invention for constructing rigid and flexible structures, control the degree of cross-linking, and finally form rigid and flexible structures that meet the design requirements of gradient degradation.

[0013] In some embodiments of the present invention, the first water-soluble cellulose derivative and the second water-soluble cellulose derivative are independently selected from one or more of the following substances: hydroxymethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxybutyl cellulose, hydroxyethyl methyl cellulose, and hydroxypropyl methyl cellulose. The first water-soluble cellulose derivative and the second water-soluble cellulose derivative can be the same or different. In specific embodiments of the present invention, the first water-soluble cellulose derivative is selected from one or more of the following substances: hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and hydroxyethyl cellulose; the second water-soluble cellulose derivative is selected from one or more of the following substances: sodium carboxymethyl cellulose and hydroxyethyl cellulose.

[0014] In some embodiments of the present invention, the viscosity of the first water-soluble cellulose derivative is 2500 - 100000 mPa·s, or 5000 - 60000 mPa·s, or 15000 - 100000 mPa·s; the viscosity of the second water-soluble cellulose derivative is 1000 - 70000 mPa·s, or 2500 - 30000 mPa·s, or 3500 - 30000 mPa·s; the viscosity test concentration is 2% for both, and the temperature is 20°C for both.

[0015] In some embodiments of the present invention, the viscosity of the first water-soluble cellulose derivative is 5000 - 60000 mPa·s, the viscosity of the second water-soluble cellulose derivative is 2500 - 30000 mPa·s, the viscosity test concentration is 2% for both, and the temperature is 20°C for both.

[0016] In some embodiments of the present invention, the first water-soluble cellulose derivative is selected from one or more of the following substances: hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, hydroxyethyl cellulose; the second water-soluble cellulose derivative is selected from one or more of the following substances: sodium carboxymethylcellulose and hydroxyethyl cellulose.

[0017] In some embodiments of the present invention, the mass ratio of the first water-soluble cellulose derivative to the second water-soluble cellulose derivative is (0.2 - 5):1.

[0018] In some embodiments of the present invention, the preparation of the gradient-degrading medical sponge further includes a hot pressing and shaping step: the sponge obtained by freeze-drying is subjected to steam treatment and then hot pressed and shaped. In an alternative embodiment, this hot pressing and shaping step can also be omitted.

[0019] In some embodiments of the present invention, it further includes irradiating and sterilizing the medical sponge after hot pressing treatment.

[0020] In some embodiments of the present invention, when the medical sponge contacts an aqueous solution, the flexible part hydrates and degrades within 12 hours to 3 days, and the rigid part hydrates and degrades within 3 to 28 days.

[0021] The application of the gradient-degrading medical sponge of the present invention as a reservoir and / or carrier for cells has the following advantages:

[0022] First, during the microfracture surgery, using the gradient-degrading medical sponge to absorb or hold the inflowing blood and wrap / attract mesenchymal stem cells (MSCs) in the bone marrow is beneficial for further developing potential tissue repair applications.

[0023] Secondly, the present invention can absorb and accommodate chondrocytes by using a medical sponge with gradient degradation. The isolated chondrocytes can be seeded onto the sponge, and the sponge loaded with chondrocytes can be implanted into the cartilage defect for cartilage repair. The present invention also uses the medical sponge to absorb and accommodate cartilage as a reservoir or carrier for cartilage. The cartilage can be from minced cartilage (such as cartilage fragments, cartilage segments) or surrounding cartilage. Without separation or cell culture, the sponge with cartilage fragments / cartilage slices or the sponge alone can be used for subchondral bone repair and cartilage defect repair.

[0024] Thirdly, the present invention uses a medical sponge with gradient degradation to absorb and accommodate early osteoblasts as a reservoir or carrier for early osteoblasts, which is beneficial for further exploring the potential applications of subchondral bone repair and cartilage defect repair.

[0025] The following further details the application of the medical sponge with gradient degradation of the present invention as a cell reservoir or carrier in conjunction with the accompanying drawings and specific embodiments. Description of the Drawings

[0026] Figure 1 Shows the total number of human mesenchymal stem cells MSC absorbed by the medical sponge with gradient degradation from the cell culture dish within 2 minutes in Example 1 of the present invention.

[0027] Figure 2 Shows the percentage of human mesenchymal stem cells MSC absorbed by the medical sponge with gradient degradation from the cell culture dish within 2 minutes in Example 1 of the present invention among the seeded cells.

[0028] Figure 3 Shows the total number of chondrocytes absorbed by the medical sponge with gradient degradation from the cell culture dish within 2 minutes in Example 2 of the present invention.

[0029] Figure 4 Shows the percentage of chondrocytes absorbed by the medical sponge with gradient degradation from the cell culture dish within 2 minutes in Example 2 of the present invention among the seeded cells.

[0030] Figure 5 Shows the total number of early osteoblasts absorbed by the medical sponge with gradient degradation from the cell culture dish within 2 minutes in Example 3 of the present invention.

[0031] Figure 6 Shows the percentage of early osteoblasts absorbed by the medical sponge with gradient degradation from the cell culture dish within 2 minutes in Example 3 of the present invention among the seeded cells. Detailed Description of the Invention

[0032] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments and the accompanying drawings. The described embodiments are only some exemplary embodiments of the present invention, rather than all embodiments. The following description of some exemplary embodiments is merely illustrative and does not constitute any limitation to the protection scope of the present invention. All other embodiments that can be obtained by those of ordinary skill in the art based on the specific embodiments recorded in the present invention without creative efforts fall within the protection scope of the present invention.

[0033] Example 1

[0034] The medical sponge used in this example is prepared based on the gradient-degrading medical sponge of Example 4 described in CN113599565B. The preparation steps are as follows:

[0035] (1) Construct a rigid structure: At room temperature, add 4 g of hydroxyethyl cellulose (2% viscosity: 100000 mPa·s, 20 °C) to 96 g of purified water, stir well to dissolve, make it into a gel state to obtain a hydroxyethyl cellulose hydrogel slurry. Adjust the pH of the obtained hydroxyethyl cellulose hydrogel slurry to 3 with 1 mol / L phosphoric acid, then add 0.001 g of glutaraldehyde solution, mix well and let stand at room temperature for 4 h to obtain a cross-linked hydroxyethyl cellulose hydrogel slurry with a 2% viscosity (20 °C) of 180000 mPa·s.

[0036] (2) Construct a flexible structure: At room temperature, disperse 0.8 g of hydroxyethyl cellulose (substitution degree of 1.8, 2% viscosity: 2500 mPa·s, 20 °C) in 20 g of absolute ethanol, add 0.01 g of glyoxal solution, after reacting for 30 minutes, dry to remove the residual solvent to obtain a hydrophobic modified hydroxyethyl cellulose powder with a substitution degree of 2.5. Then add water to 100 g, mix well and let stand at room temperature for 4 h to make a hydrophobic modified hydroxyethyl cellulose hydrogel slurry with a 2% viscosity (20 °C) of 50000 mPa·s.

[0037] (3) Construct a composite structure: Add the hydrophobic modified hydroxyethyl cellulose hydrogel slurry obtained in step (2) to the cross-linked hydroxyethyl cellulose hydrogel slurry obtained in step (1), stir at room temperature at 50 r / min for 1 h, then let stand for 2 h to defoam. Then pour it into a PETG blister box and place it in a vacuum freeze dryer to obtain a gradient-degrading medical sponge by freeze drying.

[0038] The gradient-degrading medical sponge used in Example 1 of the present invention is used to absorb and accommodate human bone marrow mesenchymal stem cells MSC. The effect verification test is as follows:

[0039] 1. MSC separation and amplification:

[0040] Bone marrow was collected from the proximal femur of patients who had received joint replacement surgery and transferred to a 50 mL centrifuge tube containing 5000 IU heparin, washed with Dulbecco's phosphate buffered saline (DPBS), and centrifuged at 150 g for 7 minutes at room temperature. Lymphocyte separation medium with a density of 1.077 g / mL (GE Healthcare Life Science, Uppsala, Sweden) was used for density gradient separation of MSCs by centrifugation at 150 g for 30 minutes at room temperature. 30 mL of DPBS was added to wash the interphase and centrifuged at 350 g for 7 minutes at room temperature, and the supernatant was discarded. The pellet was resuspended in 1 mL of FBS medium (prepared from low-glucose DMEM medium (1 g / L D-glucose, 1 mM sodium pyruvate, Sigma Aldrich), 10% fetal bovine serum FBS (Biochrom AG), 1000 IU / mL heparin (Carl Roth), 1% L-glutamine (Lonza Walkersville Inc.), 1% penicillin-streptomycin (Gibco)), transferred to a culture flask, and further cultured in an incubator at 37 °C and 5% CO2. After 24 hours and 96 hours, the medium was changed to remove non-adherent cells, and then the medium was changed twice a week. After 5-7 days, when the cell density reached 70-80%, 5 mL of Accutase cell digestion solution was used to digest the cells, digested at 37 °C for 5 minutes, counted, and 100,000 cells were re-inoculated into a new culture flask. The medium was changed twice a week, and after culturing for 5-7 days, the cells were inoculated for experiments.

[0041] 2. Culture MSCs on a six-well plate:

[0042] The MSCs were washed 3 times with 5 mL of DPBS, and 5 mL of Accutase cell digestion solution was used to digest the cells, digested at 37 °C for 5 minutes. The digested cells were transferred to a 50 mL Falcon tube, counted, and the MSCs were inoculated into each well of a six-well plate (Corning REF3516) and cultured in an incubator at 37 °C and 5% CO2 for subsequent analysis.

[0043] 3. Test the absorption capacity of the gradient-degrading medical sponge for MSC cells:

[0044] The gradient-degrading medical sponge was cut into 8 pieces and placed in the center of the wells inoculated with MSC cells. After 2 minutes, the gradient-degrading medical sponge was transferred to a new well containing fresh medium. The remaining medium was filtered through a 100-μm filter (Grainer, 542000), and 9 μL of the filtrate was aspirated and mixed with 1 μL of acridine orange / propidium iodide staining solution (Logosbio, F23001). The mixture was then used for live cell counting with a LUNA-STEM automated cell counter (Logosbio, L3001). The acridine orange / propidium iodide staining solution is a cell viability dye that causes the nuclei of live cells to fluoresce green and the nuclei of dead cells to fluoresce red. The above 10-μL mixture was loaded into a LUNA reusable slide (Logosbio, L12008), and then the slide was inserted into the counter. After manual focusing, counting was started. The number of live cells remaining in the wells that were not absorbed by the gradient-degrading medical sponge was calculated.

[0045] Data analysis was performed using SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013). The Kolmogorov-Smirnov test was used to test the normality of the data. For comparisons between two groups, Student's t-test was used for data that followed a normal distribution, and the Mann-Whitney rank sum test was used for data that did not follow a normal distribution. The analysis results are as Figure 1 and Figure 2 shown. Figure 1 Clearly shows the number of cells absorbed by the gradient-degrading medical sponge within 2 minutes and the number of remaining cells in the wells, Figure 2 shows the percentage of cells absorbed by the gradient-degrading medical sponge out of the inoculated cells. These data indicate that the gradient-degrading medical sponge has good absorption ability for MSC cells. Since only live cells were counted in this experiment, these data clearly demonstrate the occurrence of live cell absorption.

[0046] 4. Evaluate the cell viability of cells incubated in the gradient-degrading medical sponge for 7 days:

[0047] After the cell absorption experiment, the gradient-degrading medical sponge was taken out and transferred to a new well containing fresh medium, and cultured at 37 °C for 7 days. Cell viability was evaluated by microscopic examination. During the culture period, the gradient-degrading medical sponge disintegrated as expected and became a viscous gel-like substance, which hindered the evaluation of quantitative viability data using the LUNA-STEM automated cell counter. Therefore, cell viability was evaluated by microscopic examination, and the presence of a large number of live cells was verified.

[0048] From the above experimental analysis, it can be seen that the gradient-degrading medical sponge can absorb and accommodate human bone marrow mesenchymal stromal cells MSC and can serve as a reservoir or carrier for MSC.Figure 1 and Figure 2 showed that the gradient-degraded medical sponge absorbed most of the cells (95%) in the six-well plate within 2 minutes. After 7 days of culture, microscopic examination found that the gradient-degraded medical sponge showed structural disintegration as expected and turned into a viscous gel-like substance, and there were a large number of cells in the pores, most of which were live cells.

[0049] Example 2

[0050] In this example, the same gradient-degraded medical sponge as in the previous Example 1 was used, and this medical sponge was used to absorb and accommodate chondrocytes. The effect verification test is as follows:

[0051] 1. Chondrocyte isolation, amplification and culture:

[0052] Articular cartilage was collected from the proximal femur of patients undergoing joint replacement surgery. Chondrocytes were isolated overnight at 37 °C using 0.8 mg / mL Dispase-II (Roche, Germany) and 0.2 mg / mL Collagenase-XI (Sigma-Aldrich, Germany), and the cells were filtered using a 100 μm cell filter (Fisher Scientific). Monolayer cells were amplified and cultured at 37 °C and 5% CO2. The culture medium used was prepared from a 1:1 mixture of GlutaMAX / F12 medium and GlutaMAX / DMEM medium (Gibco, USA), 10% FBS supernatant, 2% PenStrep (Gibco), 1% Fungizide (Biochrom, Germany) and 0.1% L-ascorbic acid (Sigma-Aldrich). The culture medium was changed twice a week. Trypsin-EDTA solution (PAA, Germany) was used to harvest cells for experiments.

[0053] 2. Detection of the absorption capacity of the gradient-degraded medical sponge for chondrocytes:

[0054] The gradient-degrading medical sponge was cut into 8 pieces and placed in the center of the wells inoculated with chondrocytes. After 2 minutes, the gradient-degrading medical sponge was transferred to a new well containing fresh medium. The remaining medium was filtered through a 100-μm filter (Grainer, 542000), and 9 μL of the filtrate was aspirated and mixed with 1 μL of acridine orange / propidium iodide staining solution (Logosbio, F23001). The mixture was then used for live cell counting with a LUNA-STEM automatic cell counter (Logosbio, L3001). Acridine orange / propidium iodide staining solution is a cell viability dye that causes the nuclei of live cells to fluoresce green and the nuclei of dead cells to fluoresce red. The above 10-μL mixture was loaded into a LUNA reusable slide (Logosbio, L12008), and then the slide was inserted into the counter. After manual focusing, counting was started. The number of live cells remaining in the wells that were not absorbed by the gradient-degrading medical sponge was calculated.

[0055] Data analysis was performed using SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013). The Kolmogorov–Smirnov test was used to test the normality of the data. For comparisons between two groups, Student's t-test was used for data following a normal distribution, and the Mann–Whitney rank sum test was used for data not following a normal distribution. The results are as Figure 3 and Figure 4 shown. Figure 3 clearly shows the number of cells absorbed by the gradient-degrading medical sponge within 2 minutes and the number of cells remaining in the wells, Figure 4 shows the percentage of cells absorbed by the gradient-degrading medical sponge out of the inoculated cells. These data indicate that the gradient-degrading medical sponge has good absorption ability for chondrocytes. Since only live cells were counted in this experiment, these data clearly demonstrate the occurrence of live cell absorption.

[0056] 3. Evaluate the cell viability of cells incubated in the gradient-degrading medical sponge for 7 days:

[0057] After the cell absorption experiment, the gradient-degrading medical sponge was taken out and transferred to a new well containing fresh medium, and cultured at 37 °C for 7 days. Cell viability was evaluated by microscopy. During the culture period, the sponge disintegrated as expected and became a viscous gel-like substance, which hindered the evaluation of quantitative viability data using the LUNA-STEM automatic cell counter. Therefore, cell viability can be evaluated by microscopy to verify the presence of a large number of live cells.

[0058] From the above experimental analysis, it can be seen that the gradient-degrading medical sponge can absorb and accommodate chondrocytes and can serve as a reservoir or carrier for chondrocytes.

[0059] Figure 3 and Figure 4 showed that the gradient-degrading medical sponge absorbed most of the chondrocytes (95%) in the six-well plate within 2 minutes. After 7 days of culture, microscopic examination revealed that the gradient-degrading medical sponge underwent structural disintegration as expected and turned into a viscous gel-like substance, and there were a large number of cells in the pores, most of which were live cells.

[0060] Example 3

[0061] In this example, the same gradient-degrading medical sponge as in the previous Example 1 was used, and this medical sponge was used to absorb and accommodate early osteoblasts. The effect verification test is as follows:

[0062] 1. Isolation, expansion and culture of early osteoblasts:

[0063] Primary osteoblasts were derived from alveolar bone explant surgery, and alveolar bone fragments were collected from male patients during the implantation process. The alveolar bone fragments were washed with PBS and placed in a sterile culture dish. Cells were cultured using DMEM medium (Life Technologies), 10% (v / v) fetal bovine serum (Sigma-Aldrich), 2% (v / v) glutamine (Life Technologies), and 0.1 mg / mL kanamycin (Sigma-Aldrich) at 37 °C and 5% CO2. After 7 - 10 days of culturing these alveolar bone fragments, they were used as explant cultures for osteoblast growth. For cell expansion, at 37 °C, osteoblasts were digested in a PBS solution containing 0.05% trypsin / 0.02% EDTA for 5 minutes. After the cells detached and separated, three volumes of medium were added to the cell suspension to inactivate trypsin. The cell number and viability were determined using a LUNA-STEM automatic cell counter (Logosbio, L3001) and a fluorescence-based live / dead cell staining kit (acridine orange / propidium iodide stain (Logosbio, F23001)). For further experiments, the cell suspension was centrifuged at 1200 rpm for 10 minutes. The supernatant was discarded, and the pellet was resuspended in the required volume of medium and used for the experiment.

[0064] 2. Testing the absorption capacity of the gradient-degrading medical sponge for early osteoblasts:

[0065] The gradient-degrading medical sponge was cut into 8 pieces and placed in the center of the wells inoculated with early osteoblasts. After 2 minutes, the gradient-degrading medical sponge was transferred to a new well containing fresh medium. The remaining medium was filtered through a 100-μm filter (Grainer, 542000), and 9 μL of the filtrate was aspirated and mixed with 1 μL of acridine orange / propidium iodide staining solution (Logosbio, F23001). The mixture was then used for live cell counting with a LUNA-STEM automatic cell counter (Logosbio, L3001). Acridine orange / propidium iodide staining solution is a cell viability dye that can make the nuclei of live cells fluoresce green and the nuclei of dead cells fluoresce red. The above 10-μL mixture was loaded into a LUNA reusable slide (Logosbio, L12008), and then the slide was inserted into the counter. After manual focusing, the counting was started. The number of live cells remaining in the wells that were not absorbed by the gradient-degrading medical sponge was calculated.

[0066] Data analysis was performed using SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013). The Kolmogorov-Smirnov test was used to test the normality of the data. For comparisons between two groups, Student's t-test was used for data following a normal distribution, and the Mann-Whitney rank sum test was used for data not following a normal distribution. The results are shown as Figure 5 and Figure 6 shown. Figure 5 clearly shows the number of cells absorbed by the gradient-degrading medical sponge within 2 minutes and the number of remaining cells in the wells, Figure 6 shows the percentage of cells absorbed by the gradient-degrading medical sponge out of the inoculated cells. These data indicate that the gradient-degrading medical sponge has good absorption ability for early osteoblasts. Since only live cells were counted in this experiment, these data clearly demonstrate the occurrence of live cell absorption.

[0067] 3. Evaluate the cell viability of cells incubated in the gradient-degrading medical sponge for 7 days:

[0068] After the cell absorption experiment, the gradient-degrading medical sponge was taken out and placed in a new well containing fresh medium, and cultured at 37 °C for 7 days. The cell viability was evaluated by microscopic examination. During the culture period, the gradient-degrading medical sponge showed structural disintegration as expected and became a viscous gel-like substance, which hindered the evaluation of quantitative viability data using the LUNA-STEM automatic cell counter. Therefore, the cell viability could be evaluated by microscopy, verifying the presence of a large number of live cells.

[0069] From the above experimental analysis, it can be seen that the gradient-degrading medical sponge can absorb and accommodate early osteoblasts and can serve as a reservoir or carrier for early osteoblasts.

[0070] Figure 5 and Figure 6 showed that the gradient-degrading medical sponge absorbed most of the early osteoblasts (95%) in the six-well plate within 2 minutes. After 7 days of culture, microscopic examination revealed that the sponge underwent structural disintegration as expected and became a viscous gel-like substance, and there were a large number of cells in the pores, most of which were live cells.

[0071] As can be seen from the above exemplary embodiments, the present invention has successfully used the gradient-degrading medical sponge for absorbing and accommodating cells. Exemplary cells include MSCs, chondrocytes, and early osteoblasts, and may also be other types of cells. Thus, it can be known that the gradient-degrading medical sponge of the present invention can serve as a reservoir and / or carrier for cells.

[0072] The terms and phrases used in the specification of the present invention are for illustrative purposes only and do not constitute a limitation. Those skilled in the art should understand that various changes can be made to the details of the above embodiments without departing from the basic principles of the disclosed embodiments. Therefore, the protection scope of the present invention is determined only by the claims, and in the claims, unless otherwise specified, all terms should be understood in the broadest and most reasonable sense.

Claims

1. Use of a gradient-degrading medical sponge as a cell reservoir and / or carrier, wherein the preparation of the gradient-degrading medical sponge comprises the following steps: Constructing a rigid part: providing a first water-soluble cellulose derivative and crosslinking it to form a hydrogel slurry with a first viscosity, the first viscosity being 110,000 - 300,000 mPa·s, the viscosity being measured at a concentration of 2% and a temperature of 20°C; Constructing a flexible part: providing a second water-soluble cellulose derivative and crosslinking it to form a hydrogel slurry with a second viscosity, the second viscosity being 20,000 - 100,000 mPa·s, the viscosity being measured at a concentration of 2% and a temperature of 20°C; Constructing a composite structure: mixing the hydrogel slurry with the first viscosity and the hydrogel slurry with the second viscosity to form a mixed slurry; Performing vacuum freeze-drying treatment on the mixed slurry to form a sponge.

2. The use according to claim 1, wherein the cells include MSC, chondrocytes or early osteoblasts.

3. The application according to claim 1, wherein, The first viscosity is 120,000 - 210,000 mPa·s, the viscosity being measured at a concentration of 2% and a temperature of 20°C, and the second viscosity is 50,000 - 80,000 mPa·s, the viscosity being measured at a concentration of 2% and a temperature of 20°C.

4. The application according to any one of claims 1 to 3, wherein, The first water-soluble cellulose derivative and the second water-soluble cellulose derivative are independently selected from one or more of the following substances: hydroxymethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxybutyl cellulose, hydroxyethyl methyl cellulose, and hydroxypropyl methyl cellulose.

5. The application according to claim 1, wherein, The viscosity of the first water-soluble cellulose derivative is 2,500 - 100,000 mPa·s, and the viscosity of the second water-soluble cellulose derivative is 1,000 - 70,000 mPa·s, the viscosity being measured at a concentration of 2% and a temperature of 20°C for both.

6. The application according to claim 4, wherein, The viscosity of the first water-soluble cellulose derivative is 5,000 - 60,000 mPa·s, and the viscosity of the second water-soluble cellulose derivative is 2,500 - 30,000 mPa·s, the viscosity being measured at a concentration of 2% and a temperature of 20°C for both.

7. The application according to claim 1, wherein The first water-soluble cellulose derivative is selected from one or more of the following substances: hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose; the second water-soluble cellulose derivative is selected from one or more of the following substances: sodium carboxymethyl cellulose and hydroxyethyl cellulose.

8. The application according to claim 1, wherein The mass ratio of the first water-soluble cellulose derivative to the second water-soluble cellulose derivative is (0.2 - 5):

1.

9. The use according to claim 1, wherein the preparation of the gradient-degrading medical sponge further comprises a hot pressing and shaping step: subjecting the sponge obtained by freeze-drying to steam treatment and then performing hot pressing and shaping.

10. The use according to claim 1, when the medical sponge is in contact with an aqueous solution, the flexible part hydrates and degrades within 12 hours to 3 days, and the rigid part hydrates and degrades within 3 to 28 days.

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