A hydrogel for enhancing tendon-bone healing and its preparation method

Abstract

The present invention provides a hydrogel for enhancing tendon-bone healing and a preparation method thereof. The preparation method comprises: obtaining small extracellular vesicles derived from adipose-derived stem cells; adding a photoinitiator and small extracellular vesicles to a mixed solution of methacrylated hyaluronic acid and sodium alginate, and stirring evenly to obtain a SA / HA / sEVs solution; adding the SA / HA / sEVs solution to a cross-linking agent to cross-link to obtain hydrogel microfilaments; cleaning the hydrogel microfilaments, and then placing them in a mold, and repeatedly combing them with a fine-tooth comb until the hydrogel microfilaments are basically in a directional state; irradiating with an ultraviolet lamp to promote ultraviolet cross-linking of the hydrogel microfilaments to obtain the above-mentioned hydrogel for enhancing tendon-bone healing. The macroporous directional structure hydrogel functionalized with small extracellular vesicles provided by the present invention can, on the one hand, promote the repair of the supraspinatus tendon, and on the other hand, can also improve the osteoporosis of the humerus after rotator cuff repair; by simultaneously repairing bone and tendon, the hydrogel can enhance the mechanical strength of rotator cuff tendon-bone healing, and has very good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical materials, and in particular to a hydrogel for enhancing tendon-bone healing and a preparation method thereof. Background Art

[0002] With the aging of the population and the development of national fitness, the incidence of rotator cuff injuries has increased annually, currently reaching approximately 20%. Despite the continuous advancement of arthroscopic rotator cuff suturing techniques, the current retear rate after rotator cuff repair remains at 26.6-94%. Therefore, how to enhance tendon-bone healing after rotator cuff injury and reduce the retear rate after rotator cuff suturing is a hot topic in current orthopedic clinical and basic research.

[0003] The quality of rotator cuff tendon repair is an important factor affecting tendon-bone healing, and the decline in tendon quality is closely related to the occurrence of re-tear after rotator cuff repair. Osteoporosis is also an important factor affecting re-tear after rotator cuff repair. Rotator cuff injury is a degenerative disease that is closely related to age. Most patients with rotator cuff injury are elderly and therefore often have osteoporosis. Osteoporosis can affect the quality of tendon-bone healing, and its severity is also closely related to the pullout of anchors after rotator cuff repair. However, improving osteoporosis status is often overlooked in the treatment of rotator cuff injuries. Therefore, how to simultaneously promote tendon repair and bone regeneration during rotator cuff repair is the focus of promoting rotator cuff repair. However, there are currently few tissue engineering scaffolds that can simultaneously repair and improve rotator cuff tendons and osteoporosis status. Summary of the Invention

[0004] In order to overcome the defects in the prior art, the present invention provides a hydrogel that enhances tendon-bone healing and a preparation method thereof. The hydrogel has the effect of improving osteoporosis through small cell vesicles and has a directional structure, which can simultaneously promote the improvement of rotator cuff tendon and osteoporosis status, thereby enhancing the tendon-bone healing strength and reducing the rotator cuff re-tear rate.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention is to provide a method for preparing a hydrogel for enhancing tendon-bone healing, comprising the following steps:

[0007] Step 1: Obtain small extracellular vesicles derived from adipose-derived stem cells;

[0008] Step 2: Add a photoinitiator and the above-mentioned small extracellular vesicles to a mixed solution of methacrylated hyaluronic acid and sodium alginate, and stir evenly to obtain a SA / HA / sEVs solution;

[0009] Step 3: Add the SA / HA / sEVs solution to a cross-linking agent to cross-link and obtain hydrogel microfilaments;

[0010] Step 4: Clean the hydrogel microfilaments, place them in a mold, and comb them repeatedly with a fine-tooth comb until the hydrogel microfilaments are basically oriented;

[0011] Step five: irradiate with ultraviolet light to promote ultraviolet cross-linking of the hydrogel microfilaments to obtain the above-mentioned hydrogel that increases tendon-bone healing.

[0012] Furthermore, the small extracellular vesicles in step 1 are extracted from adipose-derived stem cells by gradient centrifugation; preferably, the small extracellular vesicles are prepared by the following method: when the adipose-derived stem cells in the culture dish grow to 80-90% confluence, serum-free medium is added and cultured for 48 hours; the supernatant is collected and centrifuged at 300g for 10 minutes, 2000g for 10 minutes, 10000g for 30 minutes, and then centrifuged twice at 100000g for 70 minutes to obtain the small extracellular vesicles.

[0013] Furthermore, the mixed solution of methacrylated hyaluronic acid and sodium alginate in step 2 is obtained by completely dissolving methacrylated hyaluronic acid and sodium alginate in ddH2O; in the mixed solution of methacrylated hyaluronic acid and sodium alginate, the concentration of methacrylated hyaluronic acid is 0.1-2% (m / v), preferably 0.8-1.5% (m / v), more preferably 1% (m / v); in the mixed solution of methacrylated hyaluronic acid and sodium alginate, the concentration of sodium alginate is 0.1-2% (m / v), preferably 0.8-1.5% (m / v), more preferably 1% (m / v); more preferably, in the mixed solution of methacrylated hyaluronic acid and sodium alginate, the content ratio of methacrylated hyaluronic acid and sodium alginate is 1:1.

[0014] Furthermore, the above-mentioned photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate; the volume ratio of the mixed solution of the above-mentioned methacrylate hyaluronic acid and sodium alginate to the photoinitiator is 5-10:1, preferably 9:1.

[0015] Furthermore, in step 2, small extracellular vesicles were added to a final concentration of 5×10 9 / mL-10×10 10 / mL, preferably 1×10 10 / mL.

[0016] Furthermore, the cross-linking agent is a solution containing calcium ions, preferably a calcium chloride solution, and its concentration is preferably 0.05-0.3 mol / L, more preferably 0.15-0.24 mol / L, and even more preferably 0.2 mol / L.

[0017] Furthermore, in step five, the ultraviolet light wavelength range of the ultraviolet lamp is 200-400nm, and the irradiation time is 10-30s; preferably, the ultraviolet light wavelength of the ultraviolet lamp is 365nm, and the irradiation time is 20s.

[0018] The second aspect of the present invention is to provide a hydrogel for enhancing tendon-bone healing prepared by the above preparation method.

[0019] The third aspect of the present invention is to provide the use of the above hydrogel in the preparation of a product having the effect of enhancing tendon-bone healing in osteoporotic conditions.

[0020] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0021] The macroporous directional structure hydrogel functionalized with small extracellular vesicles provided by the present invention can, on the one hand, promote the repair of the supraspinatus tendon, and on the other hand, improve the osteoporosis of the humerus after rotator cuff repair; by simultaneously repairing bone and tendon, the hydrogel can enhance the mechanical strength of rotator cuff tendon-bone healing and has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the process of synthesizing macroporous hydrogels functionalized with aligned architecture and sEVs (MHA-sEVs) in one embodiment of the present invention;

[0023] Figure 2 HE staining results show the status of tendon repair; Figure A shows the hematoxylin and eosin (HE) staining image of the tendon-bone interface when MHA-sEVs promote rotator cuff repair, and Figure B shows the semi-quantitative score of histological staining; "***" indicates P < 0.001 compared with the control group;

[0024] Figure 3 The results of picrosirius red staining demonstrate the status of tendon repair. Figure A shows a picrosirius red-stained image of the tendon-bone interface, and Figure B shows the results of a semiquantitative analysis of picrosirius red staining. "***" indicates P < 0.001 compared with the control group.

[0025] Figure 4Figure 1 shows micro-CT (SkyScan 1176, Bruker, Germany) images used to assess bone regeneration. Figure A shows micro-CT images of humeral bone quality improvement after rotator cuff repair promoted by MHA-sEVs, including coronal images (CI), sagittal images (SI), and transverse images (TI). Figure B shows the statistical results of bone volume fraction (BV / TV). Figure C shows the statistical results of trabecular number (Tb.N).

[0026] Figure 5 These are biomechanical test images of MHA-sEVs promoting rotator cuff repair; Figures AB are the biomechanical test sites; Figure C shows the maximum load test results; Figure D shows the stiffness test results; "***" indicates P < 0.001 compared with the control group. DETAILED DESCRIPTION

[0027] The present invention will be described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention. However, the following embodiments do not limit the scope of the present invention.

[0028] In the examples, conventional methods were used unless otherwise specified, and reagents used were conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0029] Example 1

[0030] This embodiment provides a hydrogel that enhances tendon-bone healing, and its preparation method comprises the following steps (refer to Figure 1 ):

[0031] 1. Obtaining small extracellular vesicles (sEVs): When adipose-derived stem cells (ADSCs) reach 80-90% confluency in a culture dish, add serum-free medium and continue culturing for 48 hours. Collect the supernatant and centrifuge it at 300g for 10 minutes, 2000g for 10 minutes, 10,000g for 30 minutes, and then centrifuge it again at 100,000g for 70 minutes to obtain sEVs.

[0032] 2. Methacrylated hyaluronic acid (HA) and sodium alginate (SA) were dissolved in double distilled water (ddH2O) (1% / 1%) and stirred on a magnetic stirrer until completely dissolved. Then, the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (volume ratio of 9:1) and sEVs (final concentration of 1×10 10 / mL) and continue stirring under light-protected conditions.

[0033] 3. Use a propulsion pump to push the SA / HA / sEVs solution into a calcium chloride solution (concentration of 0.2 mol / L) at a rate of 5 ml / min. React at room temperature for about 10 seconds, and then wash the formed hydrogel microfilaments with ddH2O.

[0034] 4. After cleaning, use a thin rod to scoop the microfilaments into the groove of the mold (groove size: 25mm*10mm*1mm), and use a fine-tooth comb to comb them in the same direction; after each combing, fold the stretched hydrogel microfilaments into the groove of the mold and comb them again until the hydrogel microfilaments are basically oriented.

[0035] 5. Then, ultraviolet light (365 nm) was used to irradiate the microfilaments for 20 seconds to promote their ultraviolet cross-linking, thereby synthesizing sEVs-functionalized macroporous oriented structure hydrogel.

[0036] Example 2

[0037] This example verifies the performance of the hydrogel for enhancing tendon-bone healing provided in Example 1. The specific experimental steps and results are as follows:

[0038] 1. An osteoporosis rat model was established by removing both ovaries of the rats: After anesthetizing the rats with sodium pentobarbital, the bilateral abdominal hair was removed, the abdominal cavity was opened, the bilateral ovaries were removed, and the wounds were sutured. Thirteen weeks after ovariectomy, the osteoporosis rat model was successfully established. Subsequently, an acute rotator cuff injury model was established in the shoulders of osteoporotic rats, and MHA-sEVs were implanted during the operation. The steps for constructing the rotator cuff injury model are as follows: After anesthetizing the rats with sodium pentobarbital, the bilateral shoulder hair was shaved. The skin of the rat shoulders was cut open, the attachment point of the supraspinatus tendon was exposed behind the trapezius muscle, and after cutting the supraspinatus tendon, the tendon was fixed to the greater tuberosity of the humerus using 4-0 tendon sutures, and then the MHA-sEVs were implanted.

[0039] Eight weeks after surgery, rats were sacrificed via an overdose of sodium pentobarbital, and the rotator cuff-humeral complex was harvested. Micro-CT (SkyScan 1176, Bruker, Germany) was used to assess bone regeneration. Bone volume fraction and trabecular thickness in the bone tunnel region were analyzed.

[0040] 3. The specimens were decalcified, dehydrated, and paraffin-fixed before being cut into 5-μm-thick paraffin sections. Hematoxylin and eosin staining and picrosirius red staining were performed to assess tendon repair. Picrosirius red staining was semi-quantitatively analyzed using Image J. Biomechanical testing of the mechanical strength of tendon-bone healing was performed using a preload of 0.1 N and a tensile speed of 10 mm / min. The ultimate load and stiffness of the specimens were analyzed.

[0041] like Figure 2 As shown in the results, MHA-sEVs applied to the tendon-bone healing model of osteoporotic rats can significantly improve the morphology of the tendon-bone interface, improve the tendon course, and promote the integration of tendon and bone; MHA-sEVs application can significantly improve the histological score of the tendon-bone interface.

[0042] like Figure 3 As shown in the results, MHA-sEVs can significantly improve the maturation of supraspinatus tendon after application in the tendon-bone healing model of osteoporotic rats.

[0043] like Figure 4 As shown in the results, MHA-sEVs applied to the tendon-bone healing model of osteoporotic rats can increase the bone volume fraction and the number of trabeculae at the attachment site of the supraspinatus tendon and improve the osteoporotic state.

[0044] like Figure 5 As shown in the results, MHA-sEVs were applied to the tendon-bone healing model of osteoporotic rats, which enhanced the biomechanical strength of tendon-bone healing by simultaneously promoting the repair of the supraspinatus tendon and increasing the bone density at the tendon attachment site.

[0045] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. It will be apparent to those skilled in the art that any equivalent modifications and substitutions to the present invention fall within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to fall within the scope of the present invention.

Claims

1. A method for preparing a hydrogel for enhancing tendon-bone healing, characterized in that: The steps include: Step 1: Obtain small extracellular vesicles derived from adipose-derived stem cells; Wherein, the small extracellular vesicles are extracted from adipose-derived stem cells by gradient centrifugation; Step 2, adding a photoinitiator and the small extracellular vesicles to a mixed solution of methacrylated hyaluronic acid and sodium alginate, and stirring uniformly to obtain a SA / HA / sEVs solution; The mixed solution of methacrylated hyaluronic acid and sodium alginate is obtained by completely dissolving methacrylated hyaluronic acid and sodium alginate in ddH2O; in the mixed solution of methacrylated hyaluronic acid and sodium alginate, the concentration of methacrylated hyaluronic acid is 0.1-2% (m / v), the concentration of sodium alginate is 0.1-2% (m / v), and the content ratio of methacrylated hyaluronic acid to sodium alginate is 1:1; Step 3, adding the SA / HA / sEVs solution to a cross-linking agent to cross-link to obtain hydrogel microfilaments; Wherein, the cross-linking agent is a solution containing calcium ions, and its concentration is 0.05-0.3 mol / L; Step 4: Clean the hydrogel microfilaments, place them in a mold, and comb them repeatedly with a fine-tooth comb until the hydrogel microfilaments are basically oriented; Step five: using ultraviolet light to irradiate and promote ultraviolet cross-linking of the hydrogel microfilaments to obtain the hydrogel that enhances tendon-bone healing.

2. The preparation method according to claim 1, characterized in that The small extracellular vesicles are prepared by the following method: when adipose stem cells grow to 80-90% confluence in a culture dish, serum-free medium is added and culture is continued for 48 hours; the supernatant is collected and centrifuged at 300g for 10 minutes, 2000g for 10 minutes, 10000g for 30 minutes, and then centrifuged twice at 100000g for 70 minutes to obtain the small extracellular vesicles.

3. The preparation method according to claim 1, characterized in that In the mixed solution of methacrylated hyaluronic acid and sodium alginate, the concentration of methacrylated hyaluronic acid is 0.8-1.5% (m / v), and the concentration of sodium alginate is 0.8-1.5% (m / v).

4. The preparation method according to claim 1, characterized in that The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone; the volume ratio of the mixed solution of methacrylate hyaluronic acid and sodium alginate to the photoinitiator is 5-10:

1.

5. The preparation method according to claim 1, characterized in that In step 2, small extracellular vesicles were added to a final concentration of 5 × 10 9 / mL-10×10 10 / mL.

6. The preparation method according to claim 1, characterized in that The cross-linking agent is a calcium chloride solution with a concentration of 0.15-0.24 mol / L.

7. The preparation method according to claim 1, characterized in that In step 5, the ultraviolet light wavelength range of the ultraviolet lamp is 200-400nm, and the irradiation time is 10-30s.

8. A hydrogel for enhancing tendon-bone healing prepared by the method according to any one of claims 1 to 7.

9. Use of the hydrogel according to claim 8 in preparing a product having the effect of enhancing tendon-bone healing in osteoporosis.

Images