Preparation method of tendon tissue scaffold based on decellularization technology
By combining freeze-thaw treatment and enzyme treatment, and optimizing parameters and conditions, the problems of incomplete cell removal and matrix structure damage in decellularization technology were solved, and a tendon tissue scaffold with excellent mechanical properties was prepared, providing an effective biological scaffold for tendon repair.
Patent Information
- Application Number
- CN202511742610.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-25
- Publication Date
- 2026-01-23
AI Technical Summary
Existing decellularization techniques, while efficiently removing cells, struggle to protect the extracellular matrix structure of tendon tissue, leading to decreased scaffold mechanical properties or incomplete cell removal, which can trigger immune responses.
A method combining freeze-thaw treatment with trypsin digestion and nuclease treatment was adopted. By optimizing freeze-thaw cycle parameters and ultrasonic treatment conditions, and by matching the types and concentrations of trypsin and nuclease, a balance between cell removal and matrix retention was achieved.
It achieves efficient cell removal, protects the structure and bioactivity of the extracellular matrix, and produces tendon tissue scaffolds with excellent mechanical properties, making them suitable for tendon repair in the field of tissue engineering.
Smart Images
Figure CN121371316A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tendon tissue scaffold preparation, specifically a method for preparing tendon tissue scaffolds based on decellularization technology. Background Technology
[0002] Tendons, as key structures connecting muscles and bones, play an indispensable role in human movement and maintaining joint stability. However, due to their poor blood supply, relatively few cells, and low metabolic activity, tendons often undergo slow natural repair processes and the quality of repair is difficult to guarantee once damaged. This can easily lead to serious consequences such as tendon adhesions, scar formation, and joint dysfunction, significantly impacting patients' quality of life. Therefore, developing effective tendon tissue repair methods has always been a research hotspot and challenge in the fields of tissue engineering and regenerative medicine.
[0003] Tissue engineering offers a promising solution for tendon repair, its core being the construction of a bio-scaffold material that mimics the structure and function of natural tendon tissue. This scaffold not only needs good biocompatibility to avoid triggering immune responses and promoting cell adhesion, proliferation, and differentiation, but also should possess suitable mechanical properties to withstand physiological stresses in vivo, providing mechanical support for the growth of new tissue. Simultaneously, the scaffold should have appropriate degradation properties, gradually degrading after fulfilling its supporting function and being replaced by new tissue, ultimately achieving tissue regeneration and functional restoration.
[0004] Among the many methods for constructing tissue-engineered tendon scaffolds, decellularization techniques have attracted significant attention due to their ability to preserve the extracellular matrix (ECM) structure of natural tendon tissue. The ECM is rich in various bioactive components such as collagen, glycosaminoglycans, and growth factors, which play crucial regulatory roles in cell growth, differentiation, and tissue regeneration. Removing cellular components from tendon tissue through decellularization reduces immunogenicity while preserving the three-dimensional structure and bioactivity of the ECM, providing an ideal microenvironment for subsequent cell implantation and tissue regeneration.
[0005] While existing decellularization techniques can achieve some degree of cell removal and extracellular matrix preservation in tendon tissue, several problems remain to be addressed. On one hand, the pursuit of efficient decellularization often leads to excessive damage to the structure and bioactivity of the extracellular matrix, resulting in decreased mechanical properties of the scaffold and insufficient mechanical support for the growth of new tissue. For example, Triton X-100 disrupts collagen cross-links, depolymerizes fiber bundles, and loses non-collagenous functional components, leading to decreased tendon mechanical strength, loss of elasticity, and residual toxicity affecting subsequent biocompatibility. On the other hand, excessive focus on preserving the extracellular matrix structure may result in incomplete cell removal, with residual cellular components potentially triggering immune responses and hindering tissue regeneration and repair. For instance, while physical methods such as freeze-thaw cycles and ultrasonic treatment cause relatively less damage to the extracellular matrix, their individual use may be insufficient to completely destroy cell structures, leading to low cell removal efficiency. Summary of the Invention
[0006] To address the aforementioned technical problems, this invention provides a method for preparing tendon tissue scaffolds based on decellularization technology. This method solves the problem in existing technologies where it is difficult to efficiently decellularize while fully protecting the extracellular matrix structure, resulting in poor scaffold mechanical properties or incomplete cell removal triggering an immune response, thus failing to achieve a perfect balance between decellularization efficiency and matrix retention.
[0007] A method for preparing a tendon tissue scaffold based on decellularization technology includes the following steps:
[0008] S1. Freeze-thaw treatment: Place the tendon fiber bundle in a centrifuge tube containing PBS, freeze in liquid nitrogen for 3-10 minutes, then thaw in a 37°C water bath for 5-15 minutes. Repeat this freeze-thaw cycle 2-5 times.
[0009] S2. Trypsin digestion and ultrasonic treatment: The frozen and thawed tendon fiber bundles were placed in 0.1%-0.5% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40kHz, 100W and 35-40℃ for 1-15 minutes. After treatment, the fiber bundles were placed in PBS and washed 2-4 times with an ultrasonic cleaner for 3-10 minutes each time.
[0010] S3. Nuclease treatment: The tendon fiber bundles digested by trypsin and sonicated were placed in a buffer solution containing 100-200 U / mL DNase I and 50-150 μg / mL RNase A, and treated with sonication at 40 kHz, 100 W, and 35-40 ℃ for 1-15 minutes. After treatment, the fiber bundles were placed in PBS and washed 2-4 times with an ultrasonic cleaner for 3-10 minutes each time, finally obtaining a tendon tissue scaffold with intact structure and complete removal of cellular components.
[0011] Preferably, the parameters for the freeze-thaw treatment are: freezing in liquid nitrogen for 4-6 minutes, thawing in a water bath at 37°C for 8-12 minutes, and repeating the cycle 3-4 times.
[0012] Preferably, the parameters for the enzyme digestion treatment are: trypsin-EDTA concentration 0.01%-0.3%, treatment time 8-12 minutes.
[0013] Preferably, the parameters for the nuclease treatment are: DNase I concentration 120-180 U / mL, RNase A concentration 80-120 μg / mL, and treatment time 5-10 minutes.
[0014] Preferably, the tendon fiber bundles are derived from cattle.
[0015] A tendon tissue scaffold is prepared by the method described above.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] By optimizing freeze-thaw cycle parameters, precisely controlling ultrasound treatment conditions, and rationally combining the types and concentrations of trypsin and nuclease, a perfect balance between decellularization efficiency and matrix retention was achieved.
[0018] During the decellularization process, it can precisely destroy cell structure and promote the release of cell contents, achieving efficient cell removal;
[0019] Meanwhile, through the synergistic effect of physical and enzymatic methods, the collagen network structure and bioactive molecules of the extracellular matrix are cleverly protected, making the prepared tendon tissue scaffold closer to natural tissue in terms of mechanical properties and biological function, providing new ideas and methods for tendon repair research and application in the field of tissue engineering. Attached Figure Description
[0020] Figure 1 This is a flowchart illustrating the preparation process of the present invention;
[0021] Figure 2 This is a comparison image of HE and DAPI staining of tissue sections before and after decellularization treatment in Example 2 of the present invention;
[0022] Figure 3 This is a SEM image of tissue sections before and after decellularization treatment in Example 2 of the present invention. Detailed Implementation
[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] like Figure 1 As shown
[0025] Example 1:
[0026] The preparation of a tendon tissue scaffold based on decellularization technology includes the following specific steps:
[0027] Freeze-thaw treatment: Take the tendon fiber bundles from cattle, place them in centrifuge tubes containing PBS, freeze them in liquid nitrogen for 3 minutes, and then thaw them in a 37°C water bath for 5 minutes. Repeat this freeze-thaw cycle twice.
[0028] Trypsin digestion and ultrasonic treatment: The frozen-thawed tendon fiber bundles were placed in 0.01% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40kHz, 100W and 35℃ for 1 minute. After treatment, the fiber bundles were placed in PBS and washed twice with an ultrasonic cleaner for 3 minutes each time.
[0029] Nuclease treatment: The tendon fiber bundles after enzyme digestion were placed in a buffer containing 100 U / mL DNase I and 50 μg / mL RNase A and treated with sonication at 40 kHz, 100 W and 35 °C for 1 minute. Afterwards, they were washed twice with PBS under the same sonication conditions, for 3 minutes each time.
[0030] Ultimately, a tendon tissue scaffold with intact structure and complete removal of cellular components is obtained.
[0031] In Example 1, the DNA content before decellularization was 161.5 ng / mg, and the residual DNA content after decellularization was 19.9 ng / mg.
[0032] Example 2:
[0033] The preparation of a tendon tissue scaffold based on decellularization technology includes the following specific steps:
[0034] Freeze-thaw treatment: Take the tendon fiber bundles from cattle, place them in centrifuge tubes containing PBS, freeze them in liquid nitrogen for 5 minutes, and then thaw them in a 37°C water bath for 10 minutes. Repeat this freeze-thaw cycle 3 times.
[0035] Trypsin digestion and ultrasonic treatment: The frozen and thawed tendon fiber bundles were placed in 0.1% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40 kHz, 100 W and 38 ℃ for 7 minutes. After treatment, the fiber bundles were placed in PBS and washed three times with an ultrasonic cleaner for 6 minutes each time.
[0036] Nuclease treatment: The tendon fiber bundles after enzyme digestion were placed in a buffer containing 150 U / mL DNase I and 100 μg / mL RNase A and treated with sonication at 40 kHz, 100 W and 37 °C for 8 minutes. After that, they were washed with PBS three times under the same sonication conditions, each time for 5 minutes.
[0037] Ultimately, a tendon tissue scaffold with intact structure and complete removal of cellular components is obtained.
[0038] like Figure 2 Image a is an HE-stained image of a tissue section before decellularization, such as... Figure 2 b is a DAPI-stained image of a tissue section before decellularization, such as... Figure 2 c is an HE-stained image of a tissue section after decellularization, such as... Figure 2 d is a DAPI-stained image of a tissue section after decellularization;
[0039] like Figure 3 a and Figure 3 Figure b shows a SEM (Scanning Electron Microscope) image of a tissue section before decellularization in Example 2. Figure 3 The scale bar is marked as 50μm. Figure 3 b. The scale bar is marked as 5μm;
[0040] like Figure 3 c and Figure 3 As shown in d, this is a SEM (Scanning Electron Microscope) image of a tissue section before decellularization in Example 2. Figure 3 The scale bar is marked as 50μm. Figure 3 The d-scale bar is marked as 5μm.
[0041] In Example 2, the DNA content before decellularization was 186.7 ng / mg, and the residual DNA content after decellularization was 17.1 ng / mg.
[0042] Example 3:
[0043] The preparation of a tendon tissue scaffold based on decellularization technology includes the following specific steps:
[0044] Freeze-thaw treatment: Take the tendon fiber bundles from cattle, place them in centrifuge tubes containing PBS, freeze them in liquid nitrogen for 10 minutes, and then thaw them in a 37°C water bath for 15 minutes. Repeat this freeze-thaw cycle 5 times.
[0045] Trypsin digestion and ultrasonic treatment: The frozen and thawed tendon fiber bundles were placed in 0.3% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40 kHz, 100 W and 40 ℃ for 15 minutes. After treatment, the fiber bundles were placed in PBS and washed 4 times with an ultrasonic cleaner for 10 minutes each time.
[0046] Nuclease treatment: The tendon fiber bundles after enzyme digestion were placed in a buffer containing 200 U / mL DNase I and 150 μg / mL RNase A and treated with sonication at 40 kHz, 100 W and 40 °C for 15 minutes. After that, they were washed with PBS four times under the same sonication conditions, 10 minutes each time.
[0047] Ultimately, a tendon tissue scaffold with intact structure and complete removal of cellular components is obtained.
[0048] In Example 3, the DNA content before decellularization was 185 ng / mg, and the residual DNA content after decellularization was 22.1 ng / mg.
[0049] Example 4:
[0050] The preparation of a tendon tissue scaffold based on decellularization technology includes the following specific steps:
[0051] Freeze-thaw treatment: Take the tendon fiber bundles from cattle, place them in centrifuge tubes containing PBS, freeze them in liquid nitrogen for 4 minutes, and then thaw them in a 37°C water bath for 8 minutes. Repeat this freeze-thaw cycle 3 times.
[0052] Trypsin digestion and ultrasonic treatment: The frozen-thawed tendon fiber bundles were placed in 0.15% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40 kHz, 100 W and 36 ℃ for 3 minutes. After treatment, the fiber bundles were placed in PBS and washed three times with an ultrasonic cleaner for 7 minutes each time.
[0053] Nuclease treatment: The tendon fiber bundles after enzyme digestion were placed in a buffer containing 120 U / mL DNase I and 70 μg / mL RNase A and treated with sonication at 40 kHz, 100 W and 36 °C for 5 minutes. Afterwards, they were washed three times with PBS under the same sonication conditions, each time for 5 minutes.
[0054] Ultimately, a tendon tissue scaffold with intact structure and complete removal of cellular components is obtained.
[0055] Example 5:
[0056] The preparation of a tendon tissue scaffold based on decellularization technology includes the following specific steps:
[0057] Freeze-thaw treatment: Take the tendon fiber bundles from cattle, place them in centrifuge tubes containing PBS, freeze them in liquid nitrogen for 7 minutes, and then thaw them in a 37°C water bath for 12 minutes. Repeat this freeze-thaw cycle 4 times.
[0058] Trypsin digestion and ultrasonic treatment: The frozen and thawed tendon fiber bundles were placed in 0.2% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40 kHz, 100 W and 36 ℃ for 4 minutes. After treatment, the fiber bundles were placed in PBS and washed twice with an ultrasonic cleaner for 5 minutes each time.
[0059] Nuclease treatment: The tendon fiber bundles after enzyme digestion were placed in a buffer containing 160 U / mL DNase I and 100 μg / mL RNase A and treated with sonication at 40 kHz, 100 W and 38 °C for 10 minutes. Afterwards, they were washed three times with PBS under the same sonication conditions, for 8 minutes each time.
[0060] Ultimately, a tendon tissue scaffold with intact structure and complete removal of cellular components is obtained.
[0061] Example 6:
[0062] The preparation of a tendon tissue scaffold based on decellularization technology includes the following specific steps:
[0063] Freeze-thaw treatment: Take the tendon fiber bundles from cattle, place them in centrifuge tubes containing PBS, freeze them in liquid nitrogen for 8 minutes, and then thaw them in a 37°C water bath for 10 minutes. Repeat this freeze-thaw cycle 4 times.
[0064] Trypsin digestion and ultrasonic treatment: The frozen and thawed tendon fiber bundles were placed in 0.3% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40 kHz, 100 W and 39 ℃ for 10 minutes. After treatment, the fiber bundles were placed in PBS and washed twice with an ultrasonic cleaner for 9 minutes each time.
[0065] Nuclease treatment: The enzyme-digested tendon fiber bundles were placed in a buffer solution containing 180 U / mL DNase I and 130 μg / mL RNase A and treated with sonication at 40 kHz, 100 W and 39 °C for 12 minutes. After that, they were washed with PBS four times under the same sonication conditions, each time for 6 minutes.
[0066] Ultimately, a tendon tissue scaffold with intact structure and complete removal of cellular components is obtained.
[0067] Experimental Example: Performance Verification of the Tendon Tissue Scaffold Prepared in This Invention
[0068] 1. Experimental Design
[0069] 1.1 Experimental Objective
[0070] This experiment aims to compare and evaluate the effectiveness of the method of the present invention (Example 2) with the traditional chemical decellularization method in treating tendon tissue through histological (HE), fluorescence staining (DAPI), and scanning electron microscopy (SEM) detection, with a focus on evaluating the efficiency of cellular component removal and the preservation of extracellular matrix (ECM) structural integrity.
[0071] 1.2 Experimental Grouping
[0072] This experiment was set up with four groups:
[0073] Experimental group: Tendon scaffold prepared using the method of the present invention (Example 2).
[0074] Comparative Example 1: Conventional chemical decellularization method using 1% Triton X-100 for 12 hours.
[0075] Comparative Example 2: Conventional chemical decellularization method using 0.5% SDS treatment for 24 hours.
[0076] Blank control group: Untreated natural bovine tendon fiber bundles.
[0077] 1.3 Sample Preparation
[0078] Each group prepares n=6 samples for subsequent testing.
[0079] 1.4 Detection Methods and Indicators
[0080] Hematoxylin-eosin (HE) staining: to observe the overall tissue structure, collagen fiber arrangement, and residual cell nuclei.
[0081] DAPI fluorescence staining: specifically displays DNA material in the cell nucleus and quantitatively assesses the presence of cell debris and nucleic acid residues.
[0082] Scanning electron microscopy (SEM): Observes the ultrastructure, collagen fiber arrangement, diameter and pore structure on the surface of tendon fibers.
[0083] 2. Experimental Results
[0084] 2.1 Experimental Results Table
[0085] Detection methods Evaluation indicators Blank control group Experimental group (Example 2) Comparative Example 1 (Triton X-100) Comparative Example 2 (SDS) HE staining cell nucleus visibility Numerous blue cell nuclei, with intact morphology Almost no cell nucleus residue A small amount of nuclear debris remains Very few cell nuclei remain Collagen structural integrity Dense structure, with neatly arranged fibers The structure is intact, with tightly packed fibers that are slightly loose. The fibrous structure is loose, and edema occurs. The fiber structure is severely damaged and extremely loose. ECM coloring Pink, even Pink, even Pink, slightly uneven Light and uneven coloring DAPI staining blue fluorescent signal Strong and dense fluorescence signal Extremely weak fluorescence signal with no specific distribution sporadic spot fluorescence signals Weak fluorescence signal, high background DNA residue quantification (ng / mg) 170 17.1 85 45 SEM detection Fiber surface structure Smooth, continuous, with a small number of cells attached. Clean, free of cell debris, with a clear fiber surface Membranous fragments adhered to the fibers, making the fiber surface blurred. Etched holes appeared on the fiber surface, resulting in structural damage. Fiber arrangement Closely arranged in parallel bundles Tightly packed, maintaining a parallel bundle structure. Loose arrangement, weakened directionality Disordered arrangement and breakage are common.
[0086] 2.2 Analysis of Experimental Results
[0087] Cell removal efficiency analysis:
[0088] HE and DAPI results showed that the experimental group (using the method of this invention) achieved the most thorough decellularization. Almost no blue cell nuclei were visible in the HE sections, the DAPI fluorescence signal was extremely weak, and the DNA quantification content was the lowest, significantly lower than the two comparative groups. This demonstrates that the combined strategy of freeze-thaw-enzyme digestion-ultrasound-assisted nuclease can efficiently and synergistically lyse and remove cells and nucleic acid components.
[0089] Comparative Example 1 (TritonX-100), as a non-ionic detergent, can dissolve cell membranes, but its ability to remove cell nuclei is limited, resulting in the presence of nucleic acid fragments.
[0090] Comparative Example 2 (SDS), as an ionic detergent, has a stronger decellularization ability than Triton X-100. However, its strong chemical action will partially degrade DNA, resulting in the absence of intact cell nuclei under HE, but DAPI can still detect fluorescence signals with a high background, indicating the presence of residual degraded nucleic acid products.
[0091] ECM structural integrity analysis:
[0092] SEM and HE results clearly showed that the experimental group preserved the ECM structure of the natural tendon to the greatest extent. Under SEM, the fiber surface was clean, and the collagen fiber bundles maintained their original parallel arrangement and smooth, continuous morphology, forming a good natural porous structure, which is conducive to cell migration and nutrient exchange.
[0093] The fibrous structure of Comparative Example 1 became edematous and loose, because Triton X-100, while damaging the cell membrane, may also partially dissolve glycoproteins and lipids in the ECM, thus disrupting the microstructure.
[0094] Comparative Example 2 showed the worst structural integrity. The potent denaturing effect of SDS, while removing cells, severely disrupted the three-dimensional structure of collagen, leading to fiber breakage, disordered arrangement, and surface pitting. This will greatly impair the mechanical properties and biocompatibility of the scaffold.
[0095] 3. Experimental Conclusions
[0096] This experiment demonstrates through systematic comparative analysis that the ultrasound-assisted composite physical-enzymatic decellularization method (Example 2) provided by this invention is significantly superior to traditional chemical reagent (Triton X-100, SDS) treatment methods in terms of both the thoroughness of cell removal and the preservation of ECM structure.
[0097] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made by those skilled in the art to the above embodiments within the scope of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing a tendon tissue scaffold based on decellularization technology, characterized in that, Includes the following steps: S1. Freeze-thaw treatment: Place the tendon fiber bundle in a centrifuge tube containing PBS, freeze in liquid nitrogen for 3-10 minutes, then thaw in a 37°C water bath for 5-15 minutes. Repeat this freeze-thaw cycle 2-5 times. S2. Trypsin digestion and ultrasonic treatment: The frozen and thawed tendon fiber bundles were placed in 0.1%-0.5% trypsin-EDTA digestion solution and treated in an ultrasonic cleaner at 40kHz, 100W and 35-40℃ for 1-15 minutes. After treatment, the fiber bundles were placed in PBS and washed 2-4 times with an ultrasonic cleaner for 3-10 minutes each time. S3. Nuclease treatment: The tendon fiber bundles digested by trypsin and sonicated were placed in a buffer solution containing 100-200 U / mL DNase I and 50-150 μg / mL RNase A, and treated with sonication at 40 kHz, 100 W, and 35-40 ℃ for 1-15 minutes. After treatment, the fiber bundles were placed in PBS and washed 2-4 times with an ultrasonic cleaner for 3-10 minutes each time, finally obtaining a tendon tissue scaffold with intact structure and complete removal of cellular components.
2. The method for preparing a tendon tissue scaffold based on decellularization technology as described in claim 1, characterized in that, The parameters for the freeze-thaw treatment are: freezing in liquid nitrogen for 4-6 minutes, thawing in a 37°C water bath for 8-12 minutes, and repeating the cycle 3-4 times.
3. The method for preparing a tendon tissue scaffold based on decellularization technology as described in claim 2, characterized in that, The parameters for the enzyme digestion treatment are: trypsin-EDTA concentration 0.01%-0.3%, treatment time 8-12 minutes.
4. The method for preparing a tendon tissue scaffold based on decellularization technology as described in claim 1, characterized in that, The parameters for the nuclease treatment are: DNase I concentration 120-180 U / mL, RNase A concentration 80-120 μg / mL, and treatment time 5-10 minutes.
5. The method for preparing a tendon tissue scaffold based on decellularization technology as described in claim 1, characterized in that, The tendon fiber bundles are derived from cattle.
6. A tendon tissue scaffold, characterized in that, Prepared by the method described in any one of claims 1-5.