Tissue repair hydrogel with bioinductive activity, its preparation method and application

Abstract

The present invention discloses a tissue repair hydrogel with bioinductive activity, its preparation method and application, belonging to the field of biomedicine. The preparation method includes the following steps: using mulberry silk as a raw material, degumming, dissolving, adding glycidyl methacrylate, mixing evenly and dialyzing to obtain a methacrylated silk fibroin solution; adding a fullerenol solution to the silk fibroin solution, mixing evenly to obtain a mixed solution of silk fibroin and fullerenol; subjecting the mixed solution of silk fibroin and fullerenol to photo-crosslinking and enzymatic crosslinking reactions in sequence to obtain a silk fibroin hydrogel composite with fullerenol. The tissue repair hydrogel with bioinductive activity constructed by the present invention has an interconnected three-dimensional porous structure, good biocompatibility, and has a certain promoting effect on osteochondral repair. The hydrogel developed by the project has simple components, rapid preparation, and good evaluation effects in various aspects, providing a novel acellular tissue engineering scaffold for the clinical treatment of osteochondral defects and having broad clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to a tissue repair hydrogel with bioinductive activity, a preparation method thereof and an application thereof. Background Art

[0002] Musculoskeletal and joint diseases have attracted the attention of the World Health Organization in the past decade, affecting the health of tens of millions of people. At present, there is still a lack of effective treatment options for osteochondral injuries in clinical practice. Osteochondral injuries involve two tissues (cartilage and subchondral bone) with different tissue characteristics and regeneration abilities. In the repair process, the loose connection between the two tissues and the asynchronous repair speed will both cause changes in joint biomechanics, affecting the long-term performance of osteochondral tissue. Therefore, the repair of osteochondral defects is quite challenging.

[0003] Currently, common methods for clinically treating osteochondral injuries, such as arthroscopic lavage and debridement, microfracture surgery, artificial joint replacement, autologous / allogeneic osteochondral transplantation, aim to relieve joint pain and symptoms, and cannot effectively promote the repair and regeneration of osteochondral tissue; moreover, these methods have great limitations, such as donor site injury, short-lasting effects, rejection reactions, etc. Therefore, the research and development of methods that can effectively promote the repair of osteochondral injuries have extremely important clinical significance and application value. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a tissue repair hydrogel with bioinductive activity, a preparation method thereof and an application thereof. The hydrogel is prepared by the cross-linking reaction of fullerol and silk fibroin, and can promote the osteogenic / chondrogenic bidirectional differentiation of endogenous bone marrow mesenchymal stem cells, effectively promoting the repair and regeneration of osteochondral defects.

[0005] The object of the present invention can be achieved by the following technical solutions:

[0006] A preparation method of a tissue repair hydrogel with bioinductive activity, comprising the following steps:

[0007] Using mulberry silk as a raw material, through degumming, dissolving, adding glycidyl methacrylate, mixing evenly and dialysis, a methacrylated silk fibroin solution is obtained;

[0008] Adding a fullerol solution to the silk fibroin solution, mixing evenly to obtain a mixed solution of silk fibroin and fullerol;

[0009] Sequentially subjecting the mixed solution of silk fibroin and fullerol to photocrosslinking and enzymatic crosslinking reactions to obtain a silk fibroin hydrogel composite with fullerol.

[0010] Optionally, in the mixed solution of silk fibroin and fullerol, the concentration of the silk fibroin solution is 2-30% (w / v).

[0011] Optionally, in the mixed solution of silk fibroin and fullerenol, the concentration of the fullerenol solution is 0.1 - 100 μg / ml.

[0012] Optionally, the photo-crosslinking process is the curing of methacrylated silk fibroin and a photoinitiator by ultraviolet light irradiation.

[0013] Optionally, the concentration of glycidyl methacrylate is 100 - 1000 mM, the concentration of the photoinitiator is 0.1 - 1% (w / v), the wavelength of the ultraviolet lamp is 200 - 400 nm, and the irradiation time is 10 - 120 s. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0014] Optionally, the enzymatic crosslinking process is catalyzed by hydrogen peroxide / horseradish peroxidase.

[0015] Optionally, the concentration of horseradish peroxidase is 10 - 100 U / ml, and the concentration of hydrogen peroxide is 0.001 - 0.1% (w / w).

[0016] Optionally, the temperature of the crosslinking reaction is 25 - 37 °C, and the time is 5 - 200 min.

[0017] The hydrogel prepared by the above preparation method.

[0018] The application of the above hydrogel in the preparation of bone and cartilage tissue repair medicaments.

[0019] Advantages of the present invention:

[0020] The tissue repair hydrogel prepared by the crosslinking reaction of fullerenol and silk fibroin in the present invention is a hydrophilic three-dimensional network structure gel, which can provide mechanical support and biological inductive activity for the bone and cartilage defect site and is suitable for the repair of bone and cartilage injuries.

[0021] The preparation process of the present invention is simple, which maximally ensures the biological activity of fullerenol; fullerenol has good biocompatibility and can effectively promote the osteogenic / chondrogenic differentiation of bone marrow mesenchymal stem cells. The silk fibroin hydrogel can effectively carry fullerenol and provide mechanical support for the defect site, thereby promoting the repair and regeneration of the bone and cartilage injury site.

[0022] After the bio-induced active tissue repair hydrogel prepared in the present invention is successively subjected to photo-crosslinking and enzymatic crosslinking reactions, its rheological properties are significantly improved, and it can better meet the mechanical property requirements for the repair of bone and cartilage injuries. Description of the Drawings

[0023] The present invention will be further described below in conjunction with the accompanying drawings.

[0024] Figure 1 It is the general morphology diagram of the tissue repair hydrogel with bioinductive activity in Example 1 of the present invention;

[0025] Figure 2 It is the electron micrograph of the tissue repair hydrogel with bioinductive activity in Example 1 of the present invention;

[0026] Figure 3 It is the rheological property diagram of the tissue repair hydrogel with bioinductive activity and the traditional photo-crosslinked silk fibroin hydrogel in Example 1 of the present invention;

[0027] Figure 4 It is the general morphology picture of the repair of rat osteochondral defect at 8 weeks in Example 2 of the present invention;

[0028] Figure 5 It is the SO staining picture of the repair of rat osteochondral defect at 8 weeks in Example 2 of the present invention. Detailed implementation manners

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] A tissue repair hydrogel with bioinductive activity is prepared by an enzymatic cross-linking reaction of 0-dimensional carbon nanomaterial fullerol and silk fibroin. Its preparation method includes the following steps:

[0032] (1) Preparation of methacrylated silk fibroin solution:

[0033] a) Degumming: Put 10 g of mulberry silk (6A grade) into 4 L of 0.02 M sodium carbonate aqueous solution, heat in a water bath at 100 °C for 30 min, wash with 2 L of pure water by shaking, repeat this process 3 times to remove sericin protein and leave silk fibroin, dry the silk fibroin at 60 °C to obtain 7 g of dried silk fibroin for standby;

[0034] b) Dissolution: Dissolve the above-mentioned dried silk fibroin at 20% (w / v) in 9.3 M lithium bromide aqueous solution, heat at 60 °C for 1 hour until the silk fibroin is dissolved to obtain a mixed solution composed of silk fibroin and a small amount of insoluble particles

[0035] c) Methacrylation: Add 424 mM glycidyl methacrylate to the above-mentioned mixed solution, and heat and stir at 60 °C and 300 rpm for 3 hours until evenly mixed;

[0036] d) Dialysis: Dialyze the mixed solution with a regenerated cellulose dialysis bag (cut-off molecular weight 12,000 - 14,000 Daltons), and dialyze 6 times with sterile pure water 10 times the volume of the mixed solution in 3 days to remove lithium bromide ions in the solution and obtain the retentate;

[0037] e) Lyophilize the retentate at -45 °C and 20 Pa for 48 hours, and store it at -80 °C for later use;

[0038] (2) Fullerol (C 60 [OH] 28 ·17H2O) is diluted with sterile pure water to a storage solution with a concentration of 1 mg / ml and stored at 4 °C.

[0039] (3) Preparation of a tissue repair hydrogel with bioinductive activity: Dilute the high-concentration methacrylated silk fibroin solution prepared by the method of (1) with sterile pure water to a final concentration of 10% (w / v) solution. Take 1 ml of the above silk fibroin solution and place it in a 1.5 ml EP tube, add 1 μl of the fullerol solution prepared by the method of (2) to obtain a silk fibroin mixed solution with a final concentration of fullerol of 1 μg / ml. Add horseradish peroxidase with a final concentration of 20 U / ml, 0.01% (w / w) hydrogen peroxide and 0.2% (w / v) photoinitiator LAP to the solution. Mix evenly and irradiate with ultraviolet light at a wavelength of 365 nm for 10 s. Place it in an oven and heat at 37 °C for 60 min to obtain a silk fibroin hydrogel composite with fullerol, that is, a tissue repair hydrogel with bioinductive activity ( Figure 1 ). The lyophilized silk fibroin hydrogel exhibits a porous three-dimensional network structure under the electron microscope ( Figure 2 ). Compared with the traditional photo-crosslinked silk fibroin hydrogel, the tissue repair hydrogel with bioinductive activity prepared by the present invention has significantly improved rheological properties after sequential photo-crosslinking and enzyme-catalyzed crosslinking reactions ( Figure 3 ), and can better meet the mechanical property requirements for osteochondral defect repair.

[0040] Example 2

[0041] A tissue repair hydrogel with bioinductive activity is prepared by the crosslinking reaction of 0-dimensional carbon nanomaterial fullerol and silk fibroin, and its application method includes the following steps:

[0042] (1) Osteochondral injury model establishment: Six to eight-week-old male Sprague-Dawley rats (weighing 200-220 g) were anesthetized. A longitudinal incision about 1 cm long was made on the right side of the patella, and the joint capsule was incised from the medial side of the patella. The patella was dislocated laterally to expose the trochlea of the femoral condyle. A 1-mm-diameter and 1-mm-deep osteochondral defect was created in the center of the trochlea using a sternal puncture needle (diameter 1 mm).

[0043] (2) Application of silk fibroin hydrogel combined with fullerol: A tissue repair hydrogel with bioinductive activity in Example 1 was filled into the osteochondral injury created in (1), 50 μl per wound. After the model was established, the patella was reduced and sutured layer by layer. After the operation, the rats were fed with standard animal feed and allowed to move freely.

[0044] (3) Evaluation of repair effect: The animals were sacrificed at 8 weeks after the operation, and samples were taken for gross and histological evaluation. Compared with the blank control group ( Figure 4 a) and the pure silk fibroin hydrogel group ( Figure 4 b), it could be seen from gross observation that the silk fibroin hydrogel group combined with fullerol ( Figure 4 c) could significantly promote the repair of osteochondral defects, and the surface cartilage of the defect site was repaired well; the results of histological sections showed that the silk fibroin hydrogel combined with fullerol could promote the repair and regeneration of the surface cartilage and subchondral bone at the osteochondral injury site, and the cell morphology, matrix secretion, and osteochondral composition were close to those of natural osteochondral tissue ( Figure 5 ).

[0045] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0046] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. A preparation method of an osteochondral repair hydrogel with bioinductive activity, characterized in that, It includes the following steps: Using mulberry silk as raw material, through degumming, dissolving, adding glycidyl methacrylate, mixing evenly and dialyzing, to obtain methacrylated silk fibroin solution; Adding fullerol solution into the said silk fibroin solution, mixing evenly to obtain a mixed solution of silk fibroin and fullerol. In the mixed solution of silk fibroin and fullerol, the concentration of the silk fibroin solution is 2 - 30% w / v, and the concentration of the fullerol solution is 0.1 - 100 μg / ml; Sequentially subjecting the mixed solution of silk fibroin and fullerol to photocrosslinking and enzymatic crosslinking reactions to obtain a silk fibroin hydrogel composite with fullerol.

2. The preparation method of the osteochondral repair hydrogel with bioinductive activity according to claim 1, characterized in that The said photocrosslinking process is the curing of methacrylated silk fibroin and a photoinitiator under ultraviolet light irradiation.

3. The preparation method of the bio-induced active osteochondral repair hydrogel according to claim 2, characterized in that, The concentration of the glycidyl methacrylate is 100 - 1000 mM, the concentration of the photoinitiator is 0.1 - 1% w / v, the wavelength of the ultraviolet lamp is 200 - 400 nm, and the irradiation time is 10 - 120 s.

4. The preparation method of the bio-induced active osteochondral repair hydrogel according to claim 2, wherein The photoinitiator is lithium 2,4,6 - trimethylbenzoyl - phenylphosphinate.

5. The preparation method of the osteo-chondral repair hydrogel with bioinductive activity according to claim 1, characterized in that, The said enzymatic crosslinking process is catalyzed by hydrogen peroxide / horseradish peroxidase.

6. The preparation method of the osteo-chondral repair hydrogel with bioinductive activity according to claim 5, characterized in that, The concentration of the horseradish peroxidase is 10 - 100 U / ml, and the concentration of hydrogen peroxide is 0.001 - 0.1% w / w.

7. A hydrogel prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the hydrogel according to claim 7 in the preparation of a repair material for osteochondral defects.

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