Fibroblast-loaded injectable hydrogel as well as preparation method and application thereof in field of myopia prevention and control
By using a carboxymethyl chitosan-oxidized hyaluronic acid injectable hydrogel loaded with fibroblasts, combined with physical compression and cell therapy, the complications and long recovery time of traditional posterior scleral reinforcement surgery have been solved, achieving minimally invasive and effective myopia control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-20
- Publication Date
- 2026-03-03
AI Technical Summary
The long-term efficacy and safety of existing posterior scleral reinforcement surgery are still under investigation. New materials and methods for treating myopia progression are being sought, as traditional surgery carries the risk of complications and has a long recovery time.
We developed an injectable hydrogel loaded with carboxymethyl chitosan-oxidized hyaluronic acid and fibroblasts. The hydrogel system is formed through a Schiff base reaction, in which fibroblasts are loaded for physical compression and physiological remodeling of the sclera, and combined with cell therapy to prevent and control myopia.
It achieves myopia control through minimally invasive surgery, strengthening the sclera structure by physically compressing and remodeling the sclera into cells, reducing the risk of complications, shortening recovery time, and providing better myopia control results.
Smart Images
Figure CN121588024A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of biomedical materials science and myopia prevention and control medicine, specifically relating to the preparation of an injectable hydrogel of carboxymethyl chitosan-oxidized hyaluronic acid loaded with fibroblasts and its application in the field of myopia prevention and control. Background Technology
[0002] Pathological myopia is a major cause of vision loss and a complex disease involving changes in the anatomy and physiological function of the posterior segment of the eye (including the sclera). Changes in the sclera during myopia progression are particularly noteworthy. As the axial length of the eye increases, the extracellular matrix of the sclera (mainly collagen) remodels, reducing collagen fiber density and subsequently altering tissue structure. The sclera thins in the posterior pole region. Posterior scleral reinforcement is an ophthalmic surgical procedure designed to prevent or slow the progression of myopia, especially high myopia and pathological myopia. Traditionally, posterior scleral reinforcement has used artificial or natural materials to strengthen the sclera. In recent years, researchers have explored novel materials, such as highly biocompatible polymers and specially treated implant-like natural tissues. Posterior scleral reinforcement has shown potential in controlling myopia progression, but its long-term efficacy and safety are still under investigation. Finding new treatment approaches and materials for the posterior scleral region has become a major focus of pathological myopia prevention and treatment in recent years. Summary of the Invention
[0003] The purpose of this invention is to address the shortcomings of existing technologies by developing an injectable hydrogel system loaded with fibroblasts for the prevention and control of myopia axial elongation. On one hand, the compression effect of the implanted hydrogel can physically reshape the sclera to shorten the axial length. On the other hand, based on the scleral remodeling mechanism of fibroblasts, the transplantation of fibroblast-loaded hydrogels provides a promising approach for therapeutic interventions to prevent or slow myopia progression. The hydrogel can adapt to the scleral surface, providing a support matrix for cell growth, thereby enhancing the strength of scleral collagen. Furthermore, the injectable hydrogel can be administered via syringe puncture. This minimally invasive method reduces surgical errors, shortens recovery time, and lowers the risk of complications associated with larger incisions.
[0004] The technical objective of this invention is achieved through the following technical solution.
[0005] An injectable hydrogel loaded with fibroblasts comprises carboxymethyl chitosan, oxidized hyaluronic acid, and fibroblasts. Carboxymethyl chitosan and oxidized hyaluronic acid form the hydrogel system via a Schiff base reaction. Fibroblasts are then loaded into the hydrogel system, resulting in a final fibroblast density of 1 × 10⁻⁶. 6 —5×10 7 cells / mL, preferably 5×10⁻⁶ 6 —1×10 7cells / mL.
[0006] The above-mentioned method for preparing injectable hydrogels is carried out according to the following steps: carboxymethyl chitosan and oxidized hyaluronic acid are uniformly dispersed in buffer solutions simulating physiological environments to form their own homogeneous solutions. Then, fibroblasts are uniformly dispersed in at least one of the two homogeneous solutions. The two homogeneous solutions are then mixed, and carboxymethyl chitosan and oxidized hyaluronic acid are gelled in situ through Schiff base reaction, and fibroblasts are loaded into the hydrogel system.
[0007] In this invention, sterile carboxymethyl chitosan is selected, and sterile oxidized hyaluronic acid is selected.
[0008] In this invention, the buffer solution simulating the physiological environment is phosphate-buffered saline (PBS) or cell culture medium.
[0009] In this invention, the cell culture medium is a complete cell culture medium, namely DMEM medium containing 10 wt% fetal bovine serum and 1 wt% penicillin / streptomycin.
[0010] In this invention, when mixing the carboxymethyl chitosan and oxidized hyaluronic acid in their respective homogeneous solutions, the mass-volume percentage of carboxymethyl chitosan is 3-5 w / v and the mass-volume percentage of oxidized hyaluronic acid is 3-5 w / v. Equal volumes of homogeneous solutions are selected for mixing.
[0011] In this invention, oxidized hyaluronic acid is prepared through the following steps: First, hyaluronic acid powder is added to deionized water and stirred to dissolve. Then, an appropriate amount of sodium periodate is added to a sodium alginate solution, and the reaction is carried out at room temperature for 3-6 hours. Ethylene glycol is then added to terminate the reaction. Finally, the reaction solution is transferred to a dialysis bag, dialyzed in deionized water for 3 days, filtered through a 220 nm filter membrane to remove bacteria, and then freeze-dried to obtain the oxidized hyaluronic acid product.
[0012] In this invention, the fibroblasts are either extracted from donated scleral tissue or directly purchased immortalized fibroblast cell lines.
[0013] In this invention, fibroblasts are cultured in vitro and grow by adhering to the surface of tissue culture polystyrene (TCP). The culture conditions are as follows: cells are cultured in complete cell culture medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin) in a high-humidity cell culture incubator at 37°C and 5% CO2. When 80% confluence is reached, the cells are digested with 0.25% trypsin for passage or preparation of fibroblast-loaded hydrogels.
[0014] The application of the injectable hydrogel loaded with fibroblasts of the present invention in the preparation of myopia prevention and control drugs and / or devices.
[0015] Compared to physical reinforcement of the posterior sclera, the remodeling of the scleral physiological structure plays a more crucial role in the development of myopia. Therefore, cell therapy offers a novel approach to preventing myopia progression, with fibroblasts being a key factor in this process. Transplanting cultured fibroblasts into the sclera triggers collagen synthesis, forming a new layer of collagen fibers. These new collagen fibers enhance the scleral structure, and this physiological remodeling process effectively reduces axial elongation and myopic displacement in myopic eyes. Compared to free cells, cells encapsulated in injectable hydrogels maintain high viability and normal function. Therefore, this invention aims to develop injectable hydrogels loaded with fibroblasts as a promising new approach for preventing myopia progression. Specifically, when used for myopia prevention, the injectable hydrogel can not only deliver fibroblasts to remodel the scleral physiological structure but also reshape the sclera through physical compression to shorten the axial length. This synergistic physical / physiological scleral remodeling effect may lead to better treatment outcomes.
[0016] Compared to existing posterior scleral reinforcement techniques, the carboxymethyl chitosan-oxidized hyaluronic acid injectable hydrogel system loaded with fibroblasts of this invention offers the dual therapeutic effects of physical compression and shaping of the sclera and cell therapy for reshaping the physiological structure of the sclera. Furthermore, the materials used are all natural polymers and their derivatives, possessing excellent biocompatibility and biodegradability, avoiding the complication risks associated with traditional non-degradable scleral reinforcement materials. As an injectable hydrogel used in minimally invasive surgery, it can also avoid the large wounds produced by traditional surgery, reduce surgical errors, and shorten recovery time, demonstrating significant practical value and promising clinical application prospects. Attached Figure Description
[0017] Figure 1 These are the 1H NMR spectra of hyaluronic acid (HA) and oxidized hyaluronic acid (HA-CHO).
[0018] Figure 2 These are digital photographs and structural diagrams of carboxymethyl chitosan-oxidized hyaluronic acid hydrogel.
[0019] Figure 3 This is a rheological property diagram of carboxymethyl chitosan-oxidized hyaluronic acid hydrogel.
[0020] Figure 4 This is an injectable demonstration photo of carboxymethyl chitosan-oxidized hyaluronic acid hydrogel.
[0021] Figure 5 This is a graph showing the results of cytotoxicity and hemolysis rate tests on carboxymethyl chitosan-oxidized hyaluronic acid hydrogel.
[0022] Figure 6This is a live / dead staining image of fibroblasts encapsulated in carboxymethyl chitosan-oxidized hyaluronic acid hydrogel (green fluorescence represents live cells; red fluorescence represents dead cells).
[0023] Figure 7 This is a demonstration of the surgical procedure and myopia prevention effect of injectable hydrogel and fibroblast-loaded injectable hydrogel in a myopic guinea pig model.
[0024] Figure 8 These are H&E staining images of the sclera of guinea pigs from different treatment groups, along with images showing the scleral thickness. Detailed Implementation
[0025] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but these examples are not intended to limit the present invention.
[0026]
[0027]
[0028] The synthesis of oxidized hyaluronic acid used in this invention is carried out according to the following steps: First, 1.5 g of hyaluronic acid is added to deionized water (200 mL) and dissolved at room temperature. Then, sodium periodate solution (0.25 M, 16.5 mL) is added to the hyaluronic acid solution, and the reaction is carried out in the dark for 3 hours. Next, ethylene glycol (1.5 mL) is added to the mixture, and the mixture is stirred for another hour to terminate the reaction. Finally, the mixture is dialyzed against deionized water for 3 days (8 kDa dialysis membrane), and the solution is filtered through a 220 nm filter membrane to remove bacteria and then freeze-dried to obtain the oxidized hyaluronic acid product. (See attached diagram) Figure 1 As shown, the successful synthesis of oxidized hyaluronic acid was characterized by 1H-NMR spectroscopy using a nuclear magnetic resonance spectrometer (500MHz, Bruker).
[0029] The preparation of a carboxymethyl chitosan-oxidized hyaluronic acid injectable hydrogel system loaded with fibroblasts was carried out according to the following steps:
[0030] First, fibroblasts cultured in vitro were digested and centrifuged to obtain a white cell pellet. Sterile carboxymethyl chitosan was dissolved in complete cell culture medium at a concentration of 5 w / v (w / v percentage, i.e., mass of sterile carboxymethyl chitosan in g / volume of complete cell culture medium in mL × 100%). Sterile oxidized hyaluronic acid was dissolved in complete cell culture medium at a concentration of 4 w / v (w / v percentage, i.e., mass of sterile oxidized hyaluronic acid in g / volume of complete cell culture medium in mL × 100%). The fibroblast pellet was resuspended homogeneously in the 4 w / v sterile oxidized hyaluronic acid solution, and then an equal volume of carboxymethyl chitosan was added and mixed thoroughly. The carboxymethyl chitosan and oxidized hyaluronic acid underwent a Schiff base reaction, resulting in rapid in-situ gelation, ultimately yielding an injectable carboxymethyl chitosan-oxidized hyaluronic acid hydrogel loaded with fibroblasts. The final fibroblast density in the hydrogel was 5 × 10⁻⁶. 6 cells / mL.
[0031] Figure 2 The inverted vial test of in-situ gelled carboxymethyl chitosan-oxidized hyaluronic acid hydrogel is shown, along with schematic diagrams of the molecular chain structures of oxidized hyaluronic acid and carboxymethyl chitosan. The aldehyde group in oxidized hyaluronic acid and the amino group in carboxymethyl chitosan can form Schiff base bonds under mild conditions, thus achieving rapid gelation.
[0032] Figure 3 The rheological properties of the carboxymethyl chitosan-oxidized hyaluronic acid (CMCS-HA-CHO) of this invention are demonstrated. As shown in Figure A, both the storage modulus (G') and loss modulus (G') of the CMCS-HA-CHO hydrogel increase over time. Therefore, the hydrogel can be injected into the sclera at a low modulus and gradually increase in modulus to compress and shape the sclera, thereby shortening the axial length of the myopic eye. As shown in Figure B, the CMCS-HA-CHO hydrogel exhibits a gel-sol-gel transition under cyclic strain scanning, indicating its injectability. Figure 4 This visually demonstrates the injectability of the carboxymethyl chitosan-oxidized hyaluronic acid hydrogel after mixing for 3 minutes, showing that the injectable properties are still maintained after mixing carboxymethyl chitosan and oxidized hyaluronic acid for 3 minutes.
[0033] Figure 5 The results showed that the carboxymethyl chitosan-oxidized hyaluronic acid hydrogel has good cell compatibility and low hemolysis rate. A and B demonstrated that the hydrogel did not show significant cytotoxicity after co-incubation with fibroblasts for 24, 48, and 72 hours, and the survival rate of fibroblasts was almost unaffected. C demonstrated that the hydrogels at different concentrations all had a hemolysis rate of less than 1%. Figure 6 This demonstrates that the fibroblasts encapsulated in the carboxymethyl chitosan-oxidized hyaluronic acid hydrogel maintain high activity, and few dead cells are observed in the field of view (red fluorescence).
[0034] Therefore, the hydrogel blended with oxidized hyaluronic acid and carboxymethyl chitosan exhibits good injectability and can maintain the activity of fibroblasts. The carboxymethyl chitosan-oxidized hyaluronic acid injectable hydrogel loaded with fibroblasts was used for myopia control, and animal experiments in a guinea pig myopia model were conducted according to the following steps:
[0035] Tenon's capsule implantation surgery was performed on myopic guinea pigs with the aid of a surgical microscope. The surgery was divided into five groups: a normal guinea pig group (Nomal), a guinea pig group with myopia modeling only (Myopia), a group with myopic guinea pigs implanted with free fibroblasts (M-Fibroblast), a group with myopic guinea pigs implanted with injectable hydrogel (M-Hydrogel), and a group with myopic guinea pigs implanted with fibroblast-loaded hydrogel (M-Fibroblast-hydrogel). All fibroblasts, injectable hydrogels, and fibroblast-loaded injectable hydrogels used in the surgery were freshly prepared. For each group, 40 μL was injected into the posterior pole of the sclera through the inferotemporal quadrant using a 1 mL syringe (the injection volume was adjusted according to individual size, ranging from 20-500 μL). The surgery did not require sutures. Postoperative ocular antibiotic treatment was necessary, with gatifloxacin instilled once daily.
[0036] Figure 7 This diagram illustrates the surgical procedures and myopia prevention effects of injectable hydrogels and fibroblast-loaded injectable hydrogels in a myopic guinea pig model. Figure A shows the surgical procedure of implanting the hydrogel into the sclera of the eyeball. Figures B and D, along with Table 1, show the effects of different treatment groups on shortening axial length and preventing myopia progression. The results show that both the injectable hydrogel group and the fibroblast-loaded injectable hydrogel group exhibited axial length shortening effects, but the fibroblast-loaded injectable hydrogel group showed better treatment efficacy and a longer effective myopia control period (at least 4 weeks). Figure 8 Table 2 shows the changes in scleral thickness in mice at week 4 under different treatment interventions. The results demonstrate that the sclera of myopic guinea pigs was thinner than that of normal guinea pigs; however, the scleral thickness in the fibroblast-loaded injectable hydrogel group significantly increased, recovering to near-normal scleral thickness. This indicates that the implantation of the fibroblast-loaded injectable hydrogel remodeled the physiological structure of the sclera in myopic guinea pigs. These results demonstrate that the fibroblast-loaded carboxymethyl chitosan-oxidized hyaluronic acid injectable hydrogel can shorten axial length and increase scleral thickness, thereby effectively controlling the progression of myopia.
[0037] Table 1. Mean axial length at various time points in different treatment groups of the myopic guinea pig model.
[0038] Average axial length (mm) week 0 week 1 week 2 week 3 week 4 Normal 8.17 8.24 8.45 8.53 8.53 Myopia 8.37 8.48 8.56 8.76 8.78 M-Fibroblast 8.34 8.44 8.45 8.58 8.72 M-Hydrogel 8.35 8.32 8.40 8.57 8.56 M-Fibroblast-hydrogel 8.35 8.20 8.15 8.25 8.34
[0039] Table 2. Mean scleral thickness at week 4 in different treatment groups of the myopic guinea pig model.
[0040]
[0041] Adjusting the process parameters according to the present invention can achieve the preparation of fibroblast-loaded oxycarboxymethyl chitosan-oxidized hyaluronic acid injectable hydrogels and their application in the field of myopia prevention and control. Testing has shown performance essentially consistent with the present invention. The present invention has been described above as exemplary. It should be noted that any simple modifications, alterations, or other equivalent substitutions that can be made by those skilled in the art without creative effort, without departing from the core of the present invention, fall within the protection scope of the present invention.
Claims
1. An injectable hydrogel loaded with fibroblasts, characterized in that, It includes carboxymethyl chitosan, oxidized hyaluronic acid, and fibroblasts. Carboxymethyl chitosan and oxidized hyaluronic acid react to form a hydrogel system, and fibroblasts are loaded into the hydrogel system.
2. The injectable hydrogel loaded with fibroblasts according to claim 1, characterized in that, The final density of fibroblasts in the hydrogel was 1×10⁻⁶. 6 —5×10 7 cells / mL.
3. The injectable hydrogel loaded with fibroblasts according to claim 1, characterized in that, The final density of fibroblasts in the hydrogel was 5 × 10⁻⁶. 6 —1×10 7 cells / mL.
4. The method for preparing the injectable hydrogel loaded with fibroblasts as described in any one of claims 1-3, characterized in that, Carboxymethyl chitosan and oxidized hyaluronic acid were uniformly dispersed in a buffer solution simulating a physiological environment to form their own homogeneous solutions. Fibroblasts were then uniformly dispersed in at least one of the two homogeneous solutions. The two homogeneous solutions were then mixed, and the carboxymethyl chitosan and oxidized hyaluronic acid were gelled in situ through the reaction, allowing the fibroblasts to be loaded into the hydrogel system.
5. The method for preparing the injectable hydrogel loaded with fibroblasts according to claim 4, characterized in that, Sterile carboxymethyl chitosan and sterile oxidized hyaluronic acid were selected.
6. The method for preparing the injectable hydrogel loaded with fibroblasts according to claim 4, characterized in that, The buffer solution that simulates the physiological environment is phosphate-buffered saline (PBS) or cell culture medium.
7. The method for preparing an injectable hydrogel loaded with fibroblasts according to claim 6, characterized in that, The cell culture medium was a complete cell culture medium, namely DMEM medium containing 10 wt% fetal bovine serum and 1 wt% penicillin / streptomycin.
8. The method for preparing the injectable hydrogel loaded with fibroblasts according to claim 4, characterized in that, When mixing carboxymethyl chitosan and oxidized hyaluronic acid in their respective homogeneous solutions, with the carboxymethyl chitosan having a mass-volume percentage of 3-5 w / v and the oxidized hyaluronic acid having a mass-volume percentage of 3-5 w / v, equal volumes of homogeneous solutions should be selected.
9. The method for preparing the injectable hydrogel loaded with fibroblasts according to claim 4, characterized in that, Fibroblasts are extracted from donated scleral tissue or directly purchased immortalized fibroblast cell lines.
10. The use of the injectable hydrogel loaded with fibroblasts as described in any one of claims 1-3 in the preparation of myopia control drugs and / or devices.