BIOACTIVE SCAFFOLD FOR INDUCTING TENDON REGENERATION, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

Provided is a method of preparing a bioactive scaffold for inducing tendon regeneration, the method includes decellularizing a fresh tendon tissue and to obtain a decellularized tendon sheet scaffold or slice scaffold, and adding ECM materials to the decellularized tendon sheet scaffold or slice scaffold.

Claims

1. A method of preparing a bioactive scaffold for inducing tendon regeneration, comprising: (1) decellularizing a fresh tendon tissue and to obtain a decellularized tendon sheet scaffold or slice scaffold; and (2) adding ECM materials to the decellularized tendon sheet scaffold or slice scaffold.

2. The method according to claim 1, wherein the preparing the decellularized tendon sheet scaffold comprises: (a1) providing a fresh tendon tissue and washing it; (a2) compressing the tendon tissue along the thickness direction to reach a compression ratio of 60% to 90% and to obtain a tendon sheet; (a3) freezing and thawing the tendon sheet and repeating the process for 4 to 6 times, each process conducted by placing the tendon sheet in liquid nitrogen for 1 to 3 minutes, followed by at 25 to 37 C. for 3 to 10 minutes; and (a4) treating the tendon sheet from the step (a3) with a nuclease and followed by washing, the nuclease treatment performed by placing the tendon sheet in a solution having DNase at a concentration of 120 to 180 IU/ml and RNase at a concentration of 80 to 120 g/ml either at room temperature or at 37 C. for 24 hours.

3. The method according to claim 1, wherein the preparing the decellularized tendon slice scaffold comprises: (b1) providing a fresh tendon tissue and washing it; (b2) freezing and thawing the tendon tissue and repeating the process for 4 to 6 times, each process carried out by placing the tendon tissue in liquid nitrogen for 1 to 3 minutes, followed by at 25 to 37 C. for 3 to 10 minutes; (b3) frozen-sectioning longitudinally the tendon tissue obtained from the step (b2) into slices of 300 to 900 m thick; and (b4) treating the slices with a nuclease and followed by washing, the nuclease treatment performed by placing the slices in a solution having DNase at a concentration of 120 to 180 IU/ml and RNase at a concentration of 80 to 120 g/ml either at room temperature or at 37 C. for 24 hours.

4. The method according to claim 1, wherein the adding ECM materials comprises: (c1) preparing a decellularized tendon gel, which comprises: (I) decellularizing, freeze-drying, and pulverizing a tendon tissue to obtain tendon powder; (II) digesting the tendon powder in a solution having 1 mg/ml of pepsin at room temperature for 24 hours and then neutralizing with a base solution; and (III) placing the digested tendon powder in a PBS solution with a 1:10 volume ratio and then in a 37 C. incubator to developing it into a decellularized tendon gel; and (c2) pasting the decellularized tendon gel onto the decellularized tendon sheet scaffold or slice scaffold prepared from the step (1) so that the tendon gel completely covers the decellularized tendon sheet scaffold or slice scaffold.

5. The method according to claim 1, wherein in the step (c1) above, the decellularizing a tendon tissue includes: (i) freezing and thawing the tendon tissue and repeating the process for 4 to 6 times, each process performed by placing the tendon tissue in liquid nitrogen for 1 to 3 minutes, followed with 3 to 10 minutes at 25 to 37 C.; (ii) frozen-sectioning longitudinally the tendon tissue into slices of 300 to 900 m thick; and (iii) placing the slices in a solution having DNase at a concentration of 120 to 150 IU/ml and RNase at a concentration of 80 to 100 g/ml in a shaker at 37 C. for 12 hours, followed by neutralizing with 0.2 N NaOH.

6. The method according to claim 1, wherein the adding ECM materials, comprises: (d1) culturing tendon cells or stem cells on the decellularized tendon sheet scaffold or slice scaffold prepared from the step (1) to form a cell-scaffold composite; and (d2) decellularizing the cell-scaffold composite after the cultured cells form dense cell sheet.

7. The method according to claim 6, wherein the stem cells are bone marrow stromal stem cells, adipose-derived stem cells, or tendon-derived stem cells.

8. The method according to claim 6, wherein 50 M vitamin C is added to the culture after the cultured cells reach 90% confluence and the culture is continued for 6 to 8 days until the cultured cells form dense cell sheet.

9. The method according to claim 6, wherein the decellularizing the cell-scaffold composite comprises placing the composite in 0.5% Triton X-100 having 20 mM ammonia water at 37 C. for 15 minutes, followed by treatment with 100 U/mL DNase at 37 C. for 2 hours.

10. The bioactive scaffold prepared according to the method of claim 1.

11. A method for treating or repairing tendon defects in a subject, comprising administering a bioactive scaffold according to the claim 10 to the subject in need thereof.

12. The method according to claim 11, wherein the bioactive scaffold is used as materials for treating or repairing tendon or ligament defects.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] These drawings illustrate certain aspects of some of the embodiments of the present invention, and should not be used to limit or define the invention.

[0045] FIG. 1 is a schematic diagram of the preparation process of a bioactive scaffold of the present invention.

[0046] FIG. 2 is a plot showing that a bioactive scaffold of the present invention covered by a tendon gel contains a variety of growth factors.

[0047] FIG. 3 contains images of H&E staining showing histology of bioactive scaffolds of the present invention after modification by the ECM of tendon-derived stem cells: Masson staining and DAPI staining confirmed that cell fractions were effectively removed by decellularization, while ECM components were retained on the surface of the decellularized tendon sheet scaffold. DTSs stand for decellularized tendon slices.

[0048] FIG. 4 is images for observation of the surface morphology of bioactive scaffolds of the present invention: The tendon-derived stem cells were cultured on the surface of the tendon slice to form a cell-scaffold composite. After decellularization, the surface of the scaffolds showed a large amount of stem cell ECM deposition. DTSs in the figure stand for decellularized tendon slices.

[0049] FIG. 5 is plots showing growth factors assay of decellularized tendon slice scaffolds modified by the ECM of tendon-derived stem cells of the present invention: ELISA quantitative results showed that the growth factor (TGF-1, VEGF and IGF-1) levels in the bioactive scaffolds after ECM modification by tendon-derived stem cells were significantly increased. In the figure, DTSs refer to decellularized tendon slices, and ECM-DTSs are bioactive scaffolds modified by ECM of tendon-derived stem cells.

[0050] FIG. 6 is plots for cytocompatibility of ECM modified decellularized tendon slice scaffolds of the present invention: SEM analysis confirmed that the surface of ECM modified bioactive scaffolds is suitable for the growth of bone marrow stromal stem cells (BMSCs) with good cell compatibility. In the figure, DTSs stand for decellularized tendon slices, and ECM-DTSs are bioactive scaffolds modified by ECM of tendon-derived stem cells.

[0051] FIG. 7 is graphs of western-blot quantitative detection of ECM protein content before and after modification by tendon-derived stem cell ECM in decellularized tendon slice scaffolds of the present invention. The results showed that the content of ECM protein components (Biglycan, Fibromodulin, Fibronectin and Vitronectin) in the bioactive scaffolds modified by ECM of tendon-derived stem cells was significantly increased. In the figure, DTSs stand for decellularized tendon slices, and ECM-DTSs are bioactive scaffolds modified by ECM of tendon-derived stem cells. * indicates a significant difference compared to the DTSs group.

[0052] FIG. 8 is images of AlamarBlue quantitatively detects the effect of bioactive scaffolds obtained from ECM of tendon-derived stem cells modified decellularized tendon slices on the proliferation of BMSCs. In the figure, DTSs refer to decellularized tendon slices, and ECM-DTSs are bioactive scaffolds modified by ECM of tendon-derived stem cells. * indicates a significant difference compared to the DTSs group.

[0053] FIG. 9 is images of live/dead cell staining results showed that BMSCs maintained higher cell viability on the ECM modified bioactive scaffold of tendon-derived stem cells. In the figure, DTSs are decellularized tendon slices, and ECM-DTSs are bioactive scaffolds modified by ECM of tendon-derived stem cells.

[0054] FIG. 10 is images for construction and surgical repair of a rabbit Achilles tendon defect model. SDFT is superficial flexor tendon, DBTs are decellularized bovine tendon sheets, and AT is Achilles tendon.

[0055] FIG. 11 is images from ultrasound observations of different groups at different time points after surgery.

[0056] FIG. 12 is images for MRI observations of different groups at different time points after surgery. B-ultrasound and MRI tests were performed on rabbit Achilles tendon at 4, 8, and 12 weeks after operation. It was found that the abnormal echo and abnormal signals in the three groups of repaired and reconstructed Achilles tendon were reduced with time. The bioactive scaffold was obviously subjected to shape change. It is gradually remodeled to the same signal as the autogenous Achilles tendon, and at the 12th week, the difference between the groups of autogenous tendon and the bioactive scaffold of this invention cannot be distinguished.

[0057] FIG. 13 is images for a general observation of different groups at different time points after surgery.

[0058] FIG. 14 is images showing histological (H&E staining) observations of different groups at different time points after surgery. Gross and histological observations showed that the scar tissue formed in the defect sites repaired by autogenous tendon and decellularized bovine tendon sheet, and the inflammatory reaction around the decellularized bovine tendon sheet was obvious at 4 weeks; the bioactive scaffold group was followed by the 12 weeks postoperatively. The defect has been reconstructed, the fiber bundles were continuous, and the inflammatory response was significantly reduced. DBTs are decellularized bovine tendon sheet scaffolds and ECM is a tendon gel.

[0059] FIG. 15 is plots for biomechanical testing of Achilles tendon repaired with bioactive scaffolds at different times after surgery. The results showed that the maximum load and stiffness of the experimental group increased gradually with time. There was no significant difference in the maximum load between the control group and the bioactive scaffold group at 12 weeks (P>0.05). The maximum load and stiffness of the tendon sheet group and the tendon gel-modified tendon sheet group were higher than those of the control group at 4 weeks (P<0.05). DBTs are decellularized bovine tendon sheet scaffolds and ECM is a tendon gel.

DETAILED DESCRIPTION

[0060] Methods are provided for preparing a bioactive scaffold for inducing tendon regeneration.

[0061] Aspects of the methods include preparing a decellularized tendon sheet scaffold or slice scaffold and preparing a decellularized tendon gel.

[0062] Before the present methods are described, it is to be understood that this invention is not limited to particular method described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

EXAMPLES

Main Materials, Reagents, and Instruments:

[0063] Fresh tendon, bone marrow stromal stem cells, tendon-derived stem cells, tendon cells;

[0064] cell culture medium, nuclease, DNA detection kit; cryostat microtome, CO.sub.2 incubator, biomechanical test system, scanning electron microscope, fluorescence microscope, freeze dryer (Christ, Germany), cryogenic ball mill (Retsch, Germany).

Statistical Method:

[0065] Statistical analysis and processing of data were performed using SPSS16.0 software package. All data were expressed as meanstandard deviation. The data satisfied with normal distribution, the variance was uniform, and the two-way comparison between groups was performed by one-way ANOVA (Sceffe method). Test standard a was set to be 0.05, ie, the difference was statistically significant at P<0.05.

Example 1, Preparing a Bioactive Scaffold for Inducing Tendon Regeneration

[0066] The bioactive decellularized tendon slice scaffold was prepared from Achilles tendon from a canine hind limb by longitudinally sectioning and was further modified by ECM of tendon-derived stem cells. The specific preparation method is as follows:

[0067] (1) providing a fresh tendon tissue and wash it; (2) freezing and thawing the tendon tissue and repeating the process for 4 to 6 times, each process carried out by placing the tendon tissue in liquid nitrogen for 1 to 3 minutes, then at 25 to 37 C. for 3 to 10 minutes; (3) frozen sectioning longitudinally the tendon tissue to obtain slices having a thickness of 300 to 900 m; (4) placing the slices in a solution having DNase at a concentration of 120 to 180 IU/ml and RNase at a concentration of 80 to 120 m/ml either at room temperature or at 37 C. for 6 to 24 hours. Finally, freeze and disinfect the thus obtained decellularized tendon slice scaffold for future use.

[0068] As shown in FIG. 1, rat tendon-derived stem cells were cultured with the tendon slice scaffold for 7 days, the culture conditions being 5% CO.sub.2 and at 37 C. When the cells reached 90% confluence, 50 M vitamin C was added for additional 8 days until the cells form dense cell sheet on the surface of the tendon slice scaffold. The whole cultured composite was then subjected to decellularization treatment, first treated with 0.5% Triton X-100 containing 20 mM ammonia water at 37 C. for 15 minutes, and then treated with 100 U/ml DNase at 37 C. for 2 hours.

[0069] That the cells were completely removed was confirmed by DNA quantification and histological observation. The ECM of tendon-derived stem cells was preserved on the tendon slice scaffold, thus achieving modification by the ECM materials. Compared to a simple tendon slice, the ECM-modified tendon slice scaffold of this invention is a better bioactive scaffold for repairing tendon defects and reconstructing tendon function.

Example 2, Preparing a Bioactive Scaffold for Inducing Tendon Regeneration

[0070] The bioactive decellularized tendon sheet scaffold was prepared from bovine Achilles tendon by compressing, decellularizing, and was further modified by the ECM of tendon-derived stem cells. The preparation method was based on the method of claim 1 of Patent No.: ZL201310636964.0. The specific preparation method is as follows:

[0071] (1) providing a fresh tendon tissue and wash it; (2) compressing the tendon tissue along the thickness direction to reach a compression ratio of 60% to 90% and obtaining a tendon sheet with a thickness of about 1.0-1.2 mm; (3) freezing and thawing the tendon sheet and repeating the process for 4 to 6 times, each process carried out by placing the tendon sheet in liquid nitrogen for 1 to 3 minutes, followed by at 25 to 37 C. for 3 to 10 minutes; and (4) placing the tendon sheet in a solution having DNase at a concentration of 120 to 180 IU/ml and RNase at a concentration of 80 to 120 m/ml either at room temperature or at 37 C. for 6 to 24 hours; then the obtained decellularized tendon sheet scaffold freeze-dried and disinfected for use.

[0072] As shown in FIG. 1, passage 3 rat tendon-derived stem cells were cultured with the decellularized tendon sheet scaffold obtained above for 7 days, the culture conditions being 5% CO.sub.2 and at 37 C. When the cells reached 90% confluence, 50 M vitamin C was added for additional 8 days until the cells form dense cell sheet on the surface of the decellularized tendon sheet scaffold. The whole cultured composite was then subjected to decellularization treatment, first treated with 0.5% Triton X-100 containing 20 mM ammonia water at 37 C. for 15 minutes, and then treated with 100 U/ml DNase at 37 C. for 2 hours.

[0073] It was confirmed by DNA quantification and histological observation that the stem cells had been completely removed, and the ECM of tendon-derived stem cells was preserved on the decellularized tendon sheet scaffold. Compared to other tendon scaffold, the ECM of tendon-derived stem cells modified decellularized tendon sheet scaffold of this invention is a better bioactive scaffold for repairing tendon defects and reconstructing tendon function.

Example 3, Preparing a Bioactive Scaffold for Inducing Tendon Regeneration

[0074] 1. Preparation Method

[0075] Bovine Achilles tendon was used to prepare a bioactive decellularized tendon sheet scaffold. Also, tendon gel was prepared. The preparation method was based on the method of claim 1 of Patent No.: ZL201310636964.0. The decellularized tendon sheet scaffold thus obtained was applied for repairing the Achilles tendon defects and reconstructing the Achilles tendon function on a rabbit. The specific preparation and use methods are as follows:

[0076] First, preparation of a decellularized tendon sheet scaffold: (1) providing a fresh tendon tissue and wash it; (2) compressing the tendon tissue along the thickness direction to reach a compression ratio of 60% to 90% and obtaining a tendon sheet with a thickness of about 1.0-1.2 mm; (3) freezing and thawing the tendon sheet and repeating the process for 4 to 6 times, each process carried out by placing the tendon sheet in liquid nitrogen for 1 to 3 minutes, followed by at 25 to 37 C. for 3 to 10 minutes; and (4) placing the tendon sheet in a solution having DNase at a concentration of 120 to 180 IU/ml and RNase at a concentration of 80 to 120 m/ml either at room temperature or at 37 C. for 6 to 24 hours; then the obtained decellularized tendon sheet scaffold freeze-dried and disinfected for use.

[0077] Next, preparation of a tendon gel: a fresh tendon tissue from rhesus macaques was decellularized by: (i) freezing and thawing the tendon tissue and repeating the process for 4 to 6 times, each process performed by placing the tendon tissue in liquid nitrogen for 1 to 3 minutes, followed by at 25 to 37 C. for 3 to 10 minutes; (ii) frozen sectioning longitudinally the tendon tissue into slices of 300-900 m thick; and (iii) placing the slices in a solution having DNase at a concentration of 120 to 150 IU/ml and RNase at a concentration of 80 to 100 g/ml in a shaker at 37 C. for 12 hours, followed by freeze-drying at 80 C. and ball milling at 20 C. to obtain tendon powder; (iv) digesting the tendon powder in a 1 mg/ml pepsin solution at room temperature for 24 hours, then neutralized with 0.2 N NaOH, and placing the digested tendon powder in a PBS solution with a 1:10 volume ratio before putting in a 37 C. incubator to develop into a tendon gel.

[0078] The decellularized tendon gel prepared above was combined with the rehydrated decellularized bovine tendon sheet scaffold also prepared above by pasting the tendon gel on the surface of the decellularized bovine tendon sheet scaffold so that the former completely covered the latter.

[0079] Adult white rabbits were used to prepare an animal model of bilateral Achilles tendon defects in the hind limb. The decellularized bovine tendon sheet scaffold was applied to repair the tendon and to reconstruct the tendon function. After the operation, gross, histological, imaging, biomechanical tests confirmed that the bioactive decellularized bovine tendon sheet scaffold showed the effect of inducing tendon regeneration and recovering the tendon function. The detailed repair experiment and results are shown in Experimental Example 1 below.

[0080] 2. Testing

[0081] Tissue lysate (FNN0071, Invitrogen; gel mass/tissue lysate volume=1 g/10 ml) was added to the tendon gel, mixed, and the mixture was placed in ice bath for 1.5 hours. The homogenate was centrifuged at 10,000 g for 20 minutes at 4 C., and the supernatant was taken for testing. An ELISA assay was performed according to the instructions of VEGF, TGF-1 and IGF-1 detection kits, respectively.

[0082] The results of ELISA showed that the tendon gel contained growth factors associated with tendon repair, VEGF, TGF-1, and IGF-1 being 1.300.53 pg/mg, 1.560.24 pg/mg, and 8.070.89 pg/mg, respectively.

[0083] The beneficial effects of the present invention will be described below by way of experimental examples.

Experimental Example 1. Properties of Bioactive Scaffolds of this Invention in Inducing Tendon Regeneration

1. Experimental Method

[0084] The bioactive scaffold prepared according to the method of Example 1 was tested for its properties as follows:

[0085] (1) Bioactive Scaffold Histological Examination

[0086] H&E staining, Masson staining, and DAPI staining were performed according to the method described in the literature: Ning L J, et al. Preparation and characterization of decellularized tendon slices for tendon tissue engineering. Journal of Biomedical Materials Research Part A, 2012: 100A: 1448-1456.

[0087] (2) Surface Morphology Observation

[0088] Surface morphology was observed using a scanning electron microscope (SEM). The specific procedure included: fixing samples to be tested (control group and experimental group) with 2.5% glutaraldehyde at 4 C. for more than 2 hours; washing in PBS for 30 minutes x 3 times; followed by alcohol-based gradient dehydration at 50%, 70%, 80%, 90%, 100% each for 15 minutes and critical point drying. Finally, scanning electron microscopy was conducted after vacuum gold spraying.

[0089] (3) Growth Factor Detection

[0090] Same as the ELISA detection method discussed above, follow the instructions of the growth factor test kits.

[0091] (4) Observation of Cell Compatibility

[0092] Same as scanning electron microscope (SEM) observation method discussed above. Samples included the decellularized tendon slice scaffolds with ECM modification by tendon-derived stem cells and scaffolds without the modification, both of them being surface-seeded with BMSCs. The sample processing method was the same as above.

[0093] (5) Western blot quantitative detection of ECM protein content changes before and after ECM modification of the decellularized tendon slice scaffolds by tendon-derived stem cells. The procedure for western-blot quantitative detection of ECM protein content in bioactive scaffolds included: extraction of total proteins in the scaffolds to be tested; total protein content of all protein samples using BCA protein concentration determination kit (following the instructions); Electrophoresis and antibody incubation of active scaffold-related proteins (Biglycan, Fibromodulin, Fibronectin and Vitronectin); Gray-scale analysis of protein bands using Gel-Pro Analyzer4 software; relative expression of target proteins in all samples was normalized by each sample's internal reference, namely, the relative expression amount of the target protein=the gray value of the target protein/the gray value of the GAPDH.

[0094] (6) AlamarBlue quantitative detection of effects on proliferation of BMSCs by the bioactive scaffolds that are modified by ECM of tendon-derived stem cells. Detection was carried out according to the method described in the literature: Ning L J, et al. Preparation and characterization of decellularized tendon slices for tendon tissue engineering. Journal of Biomedical Materials Research Part A, 2012: 100A: 1448-1456.

[0095] (7) Live/Dead Cell Staining

[0096] The surface of the decellularized tendon slice and the bioactive decellularized tendon sheet scaffold was incubated overnight in serum-free DMEM medium. BMSCs were seeded on the surface of the scaffold, and, after culturing at 5% CO.sub.2 and 37 C., live cells were stained for viability test on the first and third day: first, washing 3 times in sterile PBS; then incubated in a viability dye solution (1 ml sterile PBS+1 l calcein acetoxymethylester+1 l propidiumiodide) at 37 C. for 30 minutes in the dark. Finally, the cells were washed 3 times with sterile PBS and observed by fluorescence microscopy.

2. In Vivo Repair Experiments

[0097] The composite material prepared in Example 3 above (in which the thickness of the tendon gel covering the decellularized bovine tendon sheet scaffold was 100 to 300 m) was applied to an in vivo repair experiment as follows: 63 male adult New Zealand white rabbits were randomly selected into 3 groups: a blank group (as a control group), a simple decellularized bovine tendon sheet group (DBTs), and the composite group including the tendon gel and decellularized bovine tendon sheet (ECM+DBTs). An Achilles tendon defect model was prepared measuring 2 cm defect on the Achilles tendon (FIG. 10), and the model was tested by the treatment group to repair the Achilles defects. The blank group underwent primary repair after the Achilles tendon was cut. After the operation, B-ultrasound and MRI were performed on the 4th, 8th, and 12th week, and further histological analysis (H&E staining) and biomechanical testing were performed.

[0098] Results: B-ultrasound and MRI were performed on rabbit Achilles tendon at 4, 8, and 12 weeks after the operation. It was found that with the three groups of repaired and recovered Achilles tendon, their abnormal echo and abnormal signals decreased with time. The Achilles tendon in the composite group, after recovery, showed signals the same as that of the autogenous Achilles tendon. Any difference between the tendon repaired by the composite (having the tendon gel and decellularized bovine tendon sheet scaffold) and the autogenous tendon could not be distinguished at the 12th week (FIG. 11 and FIG. 12). Three sets of specimens were taken for general observation (FIG. 13), histological observations, and biomechanical tests. Gross and histological observations showed in the control group and simple decellularized bovine tendon sheet scaffold group, scar tissues were formed at the 4th week, and there was clear inflammatory reaction around the simple decellularized bovine tendon sheet scaffold. By contrast, in the composite group at the 12th week, the Achilles tendon defect had been repaired, the fiber bundles were continuous, and the inflammatory response was significantly reduced (FIGS. 13 and 14). The results of biomechanical tests showed that the maximum load and stiffness in the three groups increased gradually with time. There was no significant difference in the maximum load between the control group and the composite group at the 12th week (P>0.05). At the 4th week, the maximal load and stiffness in the control group were higher than those of the simple decellularized bovine tendon sheet scaffold group and the composite group (P<0.05) (FIG. 15).

3. Experimental Results

[0099] As shown in FIG. 3, H&E staining, Masson staining, and DAPI staining confirmed that the decellularization treatment effectively removed the stem cells while retaining the ECM component on the surface of the decellularized tendon slice scaffold. DTSs stand for decellularized tendon slices.

[0100] As shown in FIG. 4, tendon-derived stem cells were cultured on the surface of the tendon slice scaffold to form a cell-scaffold composite, and after decellularization treatment, a large amount of ECM deposition left by the tendon-derived stem cells was observed on the surface of the decellularized tendon slice scaffold. The DTSs in the figure refer to decellularized tendon slices.

[0101] As shown in FIG. 5, the ELISA quantitative results showed that the growth factor (TGF-1, VEGF, and IGF-1) levels in the bioactive scaffolds modified by ECM of tendon-derived stem cells were significantly increased. In the figure, DTSs stand for decellularized tendon slices, and ECM-DTSs refer to bioactive scaffolds modified by ECM of tendon-derived stem cells.

[0102] As shown in FIG. 6, SEM examination confirmed that the surface of the bioactive scaffold modified by ECM of tendon-derived stem cells is suitable for the growth of BMSCs and has good cell compatibility. In the figure, DTSs refer to decellularized tendon slices, and ECM-DTSs stand for bioactive scaffolds modified by ECM of tendon-derived stem cells.

[0103] As shown in FIG. 7, the results showed that the content of ECM protein components (Biglycan, Fibromodulin, Fibronectin, and Vitronectin) in the bioactive scaffold modified by ECM of tendon-derived stem cells was significantly increased. In the figure, DTSs refer to decellularized tendon slices, and ECM-DTSs are bioactive scaffolds modified by ECM of tendon-derived stem cells. * indicates a significant difference compared to the DTSs group.

[0104] As shown in FIG. 8, the DTSs in the figure stand for decellularized tendon slices, and the ECM-DTSs refer to bioactive scaffolds modified by the ECM of tendon-derived stem cells. * indicates a significant difference compared to the DTSs group.

[0105] As shown in FIG. 9, Live/Dead cell staining showed that BMSCs maintained higher cell viability on the bioactive scaffold that was ECM modified by tendon-derived stem cells. In the figure, DTSs refer to decellularized tendon slices, and ECM-DTSs are bioactive scaffolds modified by ECM of tendon-derived stem cells.

[0106] As shown in FIG. 10, the rabbit Achilles tendon defect model was reconstructed and surgically repaired. SDFT is superficial flexor tendon, DBTs are decellularized bovine tendon sheet, and AT is Achilles tendon.

[0107] As shown in FIG. 11, ultrasound observations of different groups at different weeks after the surgical operation; as shown in FIG. 12, Mill observations of different groups at different weeks after the surgical operation. B-ultrasound and Mill tests were performed on rabbit Achilles tendon at 4, 8, and 12 weeks after operation. It was found that the abnormality of echo and abnormal signals of the three groups of repaired and reconstructed Achilles tendon decreased with time; the composite group having the bioactive scaffold was obvious in appearance. It was gradually remodeled and had the same signal as the autogenous Achilles tendon. At the 12th week, it was impossible to distinguish the difference between the tendon tissues repaired by the autogenous tendon and by the composite group.

[0108] As shown in FIG. 13, the different groups were observed at different weeks after the surgical operation; as shown in FIG. 14, histology (H&E staining) of different groups at different weeks was observed. Gross histological observation showed images of the autogenous tendon and that in the decellularized bovine tendon sheet group (DBTs). Scar tissue was formed at the 4th week, and the inflammatory reaction around the decellularized bovine tendon sheet was visible. At the 12th week, the Achilles tendon defects in the composite group (ECM+DBTs) were repaired or had been recovered; the fiber bundles were continuous, and the inflammatory response was significantly reduced. DBTs stand for decellularized bovine tendon sheet and ECM refers to tendon gel.

[0109] As shown in FIG. 15, biomechanical test of the repaired Achilles tendons was performed at different time points after the surgical operation. The results showed that the maximum load and stiffness of the experimental group increased gradually with time. There was no significant difference in the maximum load between the control group and the composite group at the 12th week (P>0.05). At the 4th week, the maximal load and stiffness in the control group were higher than those of the simple decellularized bovine tendon sheet group and the composite group (P<0.05). DBTs are decellularized bovive tendon sheets and ECM refers to tendon gel.

[0110] In summary, the bioactive scaffold obtained by the method of this invention for inducing tendon regeneration exhibited high growth factor content, good biocompatibility, excellent in vivo repairing effects, and thus has good prospect for clinic applications.

Other Embodiments

[0111] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0112] Further, from the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.