Whole intervertebral disc decellularized tissue engineering scaffold as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological tissue engineering scaffolds, in particular to a whole intervertebral disc decellularized tissue engineering scaffold as well as a preparation method and application thereof. The method comprises the following steps: taking an intervertebral disc-bone complex of a vertebrate as a sample, and sequentially carrying out surface polishing treatment, degreasing treatment and cryopreservation rewarming treatment on the sample to obtain a pretreated sample; placing the pretreated sample in a Tris-HCl buffer solution containing a surfactant and a protease inhibitor, and carrying out oscillation treatment; putting the sample subjected to oscillation treatment into a decellularization solution, and carrying out decellularization treatment; putting the sample subjected to the decellularization treatment into a nuclease buffer solution, and carrying out nuclease treatment; and sequentially washing and freeze-drying the nuclease treated sample to obtain the whole intervertebral disc decellularized tissue engineering scaffold. Most of cell components and genetic materials are removed from the scaffold, a relatively complete extracellular matrix structure and mechanical properties are reserved, and the scaffold can be applied to the field of intervertebral disc repair and regenerative medicine.

Description

Technical Field

[0001] This invention relates to the field of bioengineering scaffold technology, and in particular to a whole intervertebral disc decellularized tissue engineering scaffold, its preparation method and application. Background Technology

[0002] Cervical spondylosis is a common orthopedic disease that seriously affects the physical and mental health of patients and has increasingly become a major global public health issue. For some patients who do not respond to conservative treatment or have severe symptoms, cervical fusion surgery is a classic surgical procedure for treating cervical spondylosis. However, this sacrifices intervertebral disc function, leading to immobility of the surgical segment and accelerated degeneration of adjacent segments. Therefore, a non-fusion technique that preserves intervertebral disc function—artificial cervical disc replacement—has emerged and achieved good short-term efficacy, showing promise as a new "gold standard" for treating this disease. However, with extended follow-up periods and an accumulation of cases, problems exposed by disc replacement, such as heterotopic ossification, loss of prosthesis mobility, accumulation of prosthesis debris, vertebral displacement and subsidence, and periprosthetic bone resorption, as well as related complications and the resulting revision surgeries, have led more and more scholars to reconsider the clinical application of disc replacement with greater caution. The reasons for this are twofold: firstly, current artificial intervertebral disc prostheses are far from perfectly replicating the structure and functional characteristics of cervical intervertebral discs, only partially achieving functional biomimicry; secondly, intervertebral disc prostheses are composed of materials such as metal, ceramics, and polyethylene, lacking any biological activity, and cannot adapt through their own growth regulation to truly integrate with surrounding tissues. Intervertebral disc regeneration tissue engineering, using seed cells as the core, tissue engineering scaffolds as carriers, and bioactive factors as inducing catalysts, holds promise for producing highly biomimetic artificial intervertebral discs, overcoming the shortcomings of existing intervertebral disc replacement technologies.

[0003] The intervertebral disc has a unique structure and complex function, consisting of an outer annulus fibrosus, upper and lower endplates, and a central nucleus pulposus. It must maintain spinal stability while ensuring spinal mobility. The pathological environment of intervertebral disc lesions is harsh, with both mechanical deterioration due to structural damage and chemical deterioration due to inflammation and nutritional deficiencies. Due to current technological limitations, intervertebral disc tissue engineering repair only treats specific areas of the disc, such as tissue-engineered scaffold repair of the annulus fibrosus and stem cell hydrogel injection repair of the nucleus pulposus. However, its effectiveness is limited and it has not been clinically applied. The main reasons are: firstly, a lack of consideration for the overall intervertebral disc; damage to any part of the disc will lead to cascading damage to other parts, therefore, repairing a single site cannot restore the overall function of the disc. Current tissue engineering repair has not yet reached a higher level of overall disc repair and reconstruction. Secondly, there is insufficient response to the pathological environment of the diseased intervertebral disc; ensuring the survival of stem cells and other seed cells in harsh pathological environments is a significant challenge. By using decellularized animal intervertebral discs, which are remarkably similar to human vertebrae, the resulting tissue-engineered scaffolds exhibit unparalleled structural biomimicry advantages over scaffolds prepared using other engineering techniques in terms of tissue structure, matrix composition, and mechanical properties. Based on this, modifying the scaffolds with anti-inflammatory and hypoxia-resistant active substances can address early-stage survival issues after tissue engineering implantation.

[0004] A major challenge in tissue-engineered intervertebral disc healing and repair lies in reconstructing the cartilage-bone interface between the disc and the vertebrae. Vertebrae possess strong self-healing capabilities (rich blood supply, active osteoblasts), while intervertebral discs (especially endplate cartilage) are avascular and have poor cell proliferation capacity, resulting in a fundamental difference in their healing abilities. Therefore, successful tissue engineering strategies must go beyond simple tissue replacement. By employing biomimetic gradient scaffolds, spatiotemporally controlled release of factors, and biphasic cell co-culture, precise simulation of the structural and functional gradients of the interface is crucial to achieving robust biointegration, rather than fibrous scar healing. Summary of the Invention

[0005] The purpose of this invention is to provide an innovative whole-disc decellularized tissue-engineered scaffold with the potential to replace existing intervertebral disc prostheses in fusion surgery, as well as its preparation method and application. The whole intervertebral disc excised in this invention retains a portion of bone tissue, allowing it to follow the classic bone healing pattern during disc replacement. Combined with osteogenic inducing factors or stem cells, this natural healing process is effectively guided and accelerated, avoiding the major problem of difficult healing of the cartilage-bone interface. Furthermore, the decellularization process successfully removes 70% of the endogenous cells, while better preserving the glycosaminoglycan content, collagen fiber structure, and mechanical properties of the intervertebral disc, and providing suitable porosity to facilitate cell seeding.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a whole intervertebral disc decellularized tissue engineering scaffold, comprising the following steps:

[0008] (1) Take the intervertebral disc-bone complex of vertebrates as a sample, and perform surface polishing, degreasing and cryopreservation thawing treatment on the sample in sequence to obtain a pre-treated sample.

[0009] (2) The pretreated sample was placed in Tris-HCl buffer containing surfactant and protease inhibitor and shaken.

[0010] (3) Place the shaken sample in a decellularization solution for decellularization.

[0011] (4) Place the decellularized sample in a nuclease buffer solution for nuclease treatment;

[0012] (5) The nuclease-treated samples were washed and freeze-dried in sequence to obtain a whole intervertebral disc decellularized tissue engineering scaffold.

[0013] Optionally, the intervertebral disc-bone complex is composed of central intervertebral disc tissue and the bone of the vertebrae above and below it; the thickness of the vertebral portions above and below the intervertebral disc-bone complex is 1 to 2 mm.

[0014] Optionally, the degreasing treatment includes: immersing the polished sample in a mixed solution of chloroform and toluene; the temperature of the degreasing treatment is 4–30°C, and the time is 4–8 h; the volume ratio of chloroform to toluene is 1–2:1.

[0015] Optionally, the cryopreservation and thawing process includes: freezing the defatted sample in liquid nitrogen and thawing it in a hypotonic buffer solution containing a protease inhibitor; the cryopreservation temperature is -196°C; the time is 3–15 min; the pH of the hypotonic buffer solution is 7.5–8; the concentration of the protease inhibitor in the hypotonic buffer solution is 1 mmol / L; and the thawing temperature is 37°C and the time is 30 min.

[0016] Optionally, the surfactant in step (2) includes Triton X-100, and the protease inhibitor includes a serine protease inhibitor; the pH of the Tris-HCl buffer is 7.2 to 8; the concentration of the surfactant in the Tris-HCl buffer is 1 to 5%, and the concentration of the protease inhibitor is 1 mmol / L; the shaking speed is 160 to 240 r / min, the temperature is 4℃, and the time is 4 to 8 h.

[0017] Optionally, the decellularization solution is a mixture of sodium dodecyl sulfate, ethylenediaminetetraacetic acid (EDTA), aprotinin, and Tris-HCl buffer; the concentration of sodium dodecyl sulfate in the decellularization solution is 0.1–1%, the concentration of EDTA is 0.1–0.5%, the concentration of aprotinin is 1–10 KIU / mL, and the concentration of Tris-HCl buffer is 10 mmol / L; the pH of the decellularization solution is 8.

[0018] The decellularization process is carried out under shaking and ultrasound, with the shaking frequency being 80-120 r / min, the ultrasound power being 10-18 kHz, and the temperature being 4℃.

[0019] Optionally, the nuclease buffer solution comprises deoxyribonuclease, ribonuclease, Tris-HCl buffer, magnesium chloride, and bovine serum albumin; the concentration of deoxyribonuclease in the nuclease buffer solution is 100–720 U / mL, the concentration of ribonuclease is 10–100 U / mL, the concentration of Tris-HCl buffer is 50 mmol / L, the concentration of magnesium chloride is 10 mmol / L, and the concentration of bovine serum albumin is 1 mg / mL; the pH value of the nuclease buffer solution is 7–7.8; the nuclease treatment temperature is 37°C, the time is 4–8 h, and the nuclease buffer solution is replaced with fresh solution every 2–4 h.

[0020] Optionally, the freeze-drying temperature is -70°C, the time is 24 to 48 hours, and the vacuum degree is 0.1 Pa.

[0021] The present invention also provides a whole intervertebral disc decellularized tissue engineering scaffold prepared by the above-described preparation method.

[0022] The present invention also provides the application of the above-mentioned whole intervertebral disc decellularized tissue engineering scaffold in the preparation of intervertebral disc repair implant materials.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The cut intervertebral disc with attached bone tissue. Currently, the most advanced intervertebral disc decellularization technology cannot completely decellularize the entire intervertebral disc (intervertebral disc-bone complex). Our technology preserves its complete physiological and mechanical structure and effectively realizes the decellularization process.

[0025] (2) Chloroform can effectively dissolve and extract hydrophobic lipids, while methanol is miscible with water and lipids, acting as a "coupling" agent to penetrate tissues, precipitate proteins, and help chloroform better contact lipids in tissues, thereby extracting them. The combination of the two can very efficiently dissolve various lipids in cell membranes and adipocytes into the organic phase, thereby achieving deep degreasing and avoiding the influence of lipids on the penetrability and elution efficiency of the decellularization solution during the decellularization process.

[0026] (3) We used liquid nitrogen repeated freeze-thaw technology to reduce the mechanical damage caused by the formation of a large number of ice crystals during the freeze-thaw process by reducing the freezing time and the method of rewarming in a 37°C water bath. In particular, we accelerated the rewarming time and avoided the damage caused by large ice crystals formed by Ostwald ripening. The small ice crystals formed by immersion in liquid nitrogen can puncture the cell membrane and maximize cell lysis. This "fast freeze + fast thaw" mode inhibits and destroys cell-matrix adhesion, which promotes the removal of cell debris and protects the extracellular matrix.

[0027] (4) For whole intervertebral disc decellularization, in addition to physical methods, we also used chemical reagents + enzyme-linked methods to increase elution efficiency. For example, using Triton X-100 decellularization effectively removes cells while maximally preserving the structure, components, and bioactivity of the natural ECM, with a relatively gentle mode of action. The combination of SDS and EDTA is a strategy to achieve ultimate decellularization through a powerful chemical synergy: the ionic detergent SDS is responsible for thoroughly dissolving the cell membrane and emulsifying the contents, while the chelating agent EDTA breaks down intercellular connections, disintegrates cell structure, and enhances permeability by stripping divalent ions from the tissue. This approach can remove cell nuclei and intracellular substances with extremely high efficiency, significantly reducing the immunogenicity of natural tissues. In addition, nuclease decellularization can accurately and efficiently remove residual nucleic acid fragments, greatly reducing immunogenicity, while perfectly preserving the natural structure and function of the extracellular matrix. It is an indispensable "refining" step in the high-quality decellularization process. Although the combined use of multiple reagents can cause significant damage to the physical structure and biochemical components (such as glycosaminoglycans) of the extracellular matrix (ECM) and carries a high risk of reagent residue, the larger and more intact whole intervertebral discs are denser and require a more penetrating strategy. To address this, key factors such as reagent concentration, ratio, reaction time, temperature, treatment method, and enzyme activity have been repeatedly verified through numerous experiments to ensure their reasonable range, thus avoiding significant loss of the original performance of the biological tissue scaffold due to excessive elution.

[0028] (5) The whole intervertebral disc scaffold prepared by freeze-drying technology successfully preserved its natural three-dimensional, highly interconnected hierarchical porous structure. This structural characteristic not only greatly promotes the efficient transport of nutrients and metabolic waste, but also provides a crucial physical space and surface microenvironment for cell migration, proliferation, and extracellular matrix secretion. At the same time, the excellent hydrophilicity imparted to the scaffold by the freeze-drying process significantly enhances its body fluid adsorption capacity, ensuring early and rapid cell adhesion and spread, thus laying a solid and reliable physicochemical foundation for subsequent in-depth biocompatibility evaluation, cell reimplantation, and even tissue regeneration research. Attached Figure Description

[0029] Figure 1 Representative physical images of the anteroposterior diameter (a), lateral diameter (b), and height (c) of a whole intervertebral disc decellularized scaffold that has not undergone lyophilization.

[0030] Figure 2 In the diagram, a is a HE staining image of each component of the scaffold after treatment in Example 2 under 4x (objective); b is a HE staining image of each component of the scaffold after treatment in Example 2 under 40x (objective); c is a quantitative analysis diagram of HE staining.

[0031] Figure 3 The graphs show the compressive mechanical properties (MTS test) of the decellularized scaffold group and the control group.

[0032] Figure 4 Representative water absorption kinetic curves of lyophilized decellularized scaffolds in different solutions (PBS buffer / deionized water / DMEM medium);

[0033] Figure 5 A statistical graph showing the changes in DNA content at different sites (nucleus pulposus, annulus fibrosus, and cartilaginous endplate) of the decellularized scaffold (experimental group) compared to the control group;

[0034] Figure 6 A statistical graph showing the changes in GAG content in different parts (nucleus pulposus, annulus fibrosus, and cartilaginous endplate) of the decellularized scaffold (experimental group) compared to the control group;

[0035] Figure 7 In the figure, a is the COL1 staining map of the annulus fibrosus and nucleus pulposus of the scaffold; b is the COL2 staining map of the annulus fibrosus and nucleus pulposus of the scaffold; c is the quantitative statistical analysis of the staining results of figures a and b.

[0036] Figure 8 Sirius red staining images of the various components of the scaffold (annulus fibrosus, nucleus pulposus, cartilaginous endplate, and vertebrae). Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] All raw materials used in this invention can be obtained commercially or prepared using existing technologies.

[0043] This invention provides a method for preparing a whole intervertebral disc decellularized tissue engineering scaffold, comprising the following steps:

[0044] (1) Take the intervertebral disc-bone complex of vertebrates as a sample, and perform surface polishing, degreasing and cryopreservation thawing treatment on the sample in sequence to obtain a pre-treated sample.

[0045] (2) The pretreated sample was placed in Tris-HCl buffer containing surfactant and protease inhibitor and shaken.

[0046] (3) Place the shaken sample in a decellularization solution for decellularization.

[0047] (4) Place the decellularized sample in a nuclease buffer solution for nuclease treatment;

[0048] (5) The nuclease-treated samples were washed and freeze-dried in sequence to obtain a whole intervertebral disc decellularized tissue engineering scaffold.

[0049] Step (1) of this invention is to use a high-speed micro bone saw or a sharp bone chisel to cut off the adjacent vertebrae along the middle of the vertebral body to obtain an intervertebral disc-bone complex sample composed of the central intervertebral disc tissue and the bone of the vertebrae above and below it. The sample is then subjected to surface polishing, degreasing and cryopreservation thawing to obtain a pre-treated sample.

[0050] In some embodiments of the present invention, the method for in-situ acquisition of the entire intervertebral disc is as follows:

[0051] Step 1: Specimen screening and preliminary processing. From commercially available fresh oxtail, the intervertebral space is identified based on anatomical landmarks. Using a sterile scalpel and bone shears, a complete kinetic unit containing the target intervertebral disc and the bone of the adjacent upper and lower vertebral bodies is cut off and immediately placed in pre-cooled PBS or physiological saline to keep the tissue moist.

[0052] Step 2: Detailed Dissection and Soft Tissue Removal. The obtained motor unit specimen is placed on a sterile operating table. Using fine surgical instruments (such as tissue scissors, forceps, and periosteal elevator), the muscles, fascia, fat, ligaments, and other attached connective tissues on the surface of the unit are sharply separated and thoroughly removed to fully expose the annulus fibrosus, endplate, and vertebral body bone surface of the intervertebral disc.

[0053] Step 3: Unit Segmentation and Target Tissue Delineation. Using a high-speed micro bone saw (or a sharp bone chisel), carefully cut adjacent vertebrae along the middle of the vertebral body to obtain a "Disc-Bone Complex" sample composed of the central intervertebral disc tissue and the bone of the vertebrae above and below it. The operation must be performed in a low-temperature environment to avoid mechanical damage to the annulus fibrosus and bony structure.

[0054] Step 4: Target Tissue Separation and Weighing Recording (if further processing is required) The above complex can be further dissected to separate the complete intervertebral disc tissue containing cartilage and adjacent cancellous bone blocks. The obtained tissues are weighed, measured, and photographed for subsequent calculation of the amount and efficiency evaluation of the decellularization reagent.

[0055] Step 5: Decellularization Pretreatment and Temporary Storage. Immediately immerse the final target tissue (intact intervertebral disc-bone complex, or separated intervertebral disc and bone fragments) in pre-cooled PBS to remove blood and free cell debris. After completion, proceed to the next step.

[0056] In some implementations, the thickness of the upper and lower vertebrae harvested should be 1 to 2 mm.

[0057] In some implementations, the resulting intervertebral disc-bone complex should be adapted to the size of the corresponding intervertebral disc in the spine of humans and other mammals.

[0058] In some embodiments of the present invention, the sample needs to be washed before polishing. The washing is performed by stirring the sample with PBS solution at 4°C for 1 hour.

[0059] In some embodiments of the present invention, the polishing process involves using sandpaper to smooth the surface of the upper and lower vertebrae.

[0060] In this invention, the degreasing treatment includes: immersing the polished sample in a mixed solution of chloroform and toluene to ensure full contact between the sample and the solution; after completion, washing three times with PBS solution; the temperature of the degreasing treatment is 4–30°C, for example, 4°C, 10°C, 15°C, 20°C, 25°C, or 30°C; the time is 4–8 hours, for example, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours; the volume ratio of chloroform to toluene is 1–2:1, for example, 1:1 or 2:1.

[0061] In some embodiments of the present invention, after the degreasing process is completed, the product is washed with distilled water and anhydrous ethanol for 1 hour each.

[0062] In this invention, the cryopreservation and thawing process includes: freezing the defatted sample in liquid nitrogen and thawing it in a hypotonic buffer solution containing a protease inhibitor; the cryopreservation temperature is -196°C; the time is 3 to 15 minutes, for example, 3 minutes, 5 minutes, 10 minutes, or 15 minutes; the pH of the hypotonic buffer solution is 7.5 to 8, for example, 7.5, 7.6, 7.7, 7.8, 7.9, or 8; the concentration of the protease inhibitor in the hypotonic buffer solution is 1 mmol / L; and the thawing temperature is 37°C and the time is 30 minutes.

[0063] In some embodiments of the present invention, the rewarmed sample needs to be washed with PBS containing protease inhibitors for 10 minutes, and the washing cycle is repeated 3 times.

[0064] In one embodiment of the present invention, after the sample cryopreservation and thawing process is completed, the sample needs to be washed with distilled water, cleaned with anhydrous ethanol, filtered for 30 minutes, and then naturally dried for later use.

[0065] In another embodiment of the present invention, after the sample cryopreservation and thawing process is completed, the sample is washed with distilled water and then subjected to gradient dehydration treatment with ethanol.

[0066] Specifically: First, wash the sample with distilled water, and then dehydrate it with ethanol at concentration gradients of 30%, 50%, 70%, and 99%, with each concentration of ethanol treated for no less than 30 minutes and the total dehydration time no less than 2 hours.

[0067] Step (2) of this invention involves adding a protease inhibitor to a Tris-HCl buffer containing a surfactant, placing the sample from the previous reaction into the buffer, and continuously shaking it on a shaker. After the reaction is complete, the sample is rinsed with distilled water for 3 hours.

[0068] In this invention, the surfactant in step (2) includes Triton X-100, and the protease inhibitor includes a serine protease inhibitor; the concentration of the Tris-HCl buffer is 10 mmol / L; the pH of the Tris-HCl buffer is 7.2 to 8, for example, 7.2, 7.5, 7.6, 7.8 or 8; the concentration of the surfactant in the Tris-HCl buffer is 1 to 5%, for example, 1%, 2%, 3%, 4% or 5%, and the concentration of the protease inhibitor is 1 mmol / L; the shaking speed is 160 to 240 r / min, for example, 160 r / min, 180 r / min, 200 r / min, 220 r / min or 240 r / min; the temperature is 4℃, and the time is 4 to 8 h.

[0069] Step (3) of this invention is to place the sample after shaking treatment in a decellularization solution for decellularization treatment, and after decellularization treatment, wash the sample with 0.5mol / L potassium chloride and distilled water for 30 min, soak it in anhydrous ethanol solution for 30 min, and let it evaporate naturally in a ventilated place for 30 min.

[0070] In this invention, the decellularization solution is a mixture of sodium dodecyl sulfate, ethylenediaminetetraacetic acid (EDTA), aprotinin, and Tris-HCl buffer. The concentration of sodium dodecyl sulfate in the decellularization solution is 0.1–1%, for example, 0.1%, 0.5%, or 1%. The concentration of EDTA is 0.1–0.5%, for example, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. The concentration of aprotinin is 1–10 KIU / mL, for example, 1 KIU / mL, 2 KIU / mL, 3 KIU / mL, 4 KIU / mL, 5 KIU / mL, 6 KIU / mL, 7 KIU / mL, 8 KIU / mL, 9 KIU / mL, or 10 KIU / mL. The concentration of Tris-HCl buffer is 10 mmol / L. The pH of the decellularization solution is 8.

[0071] The samples were treated in a decellularized solution at 4°C for 24 hours, during which time ultrasonic elution was performed every 5 hours. The ultrasonic elution was carried out under shaking and ultrasound. The shaking frequency was 80-120 r / min, for example, 80 r / min, 90 r / min, 100 r / min, 110 r / min or 120 r / min, etc., and the ultrasound power was 10-18 kHz, for example, 10 kHz, 11 kHz, 12 kHz, 13 kHz, 14 kHz, 15 kHz, 16 kHz, 17 kHz or 18 kHz, etc.

[0072] Step (4) of the present invention involves immersing the sample treated in step (3) in a 37°C Tris buffer containing deoxyribonuclease and ribonuclease. The buffer is composed of Tris-HCl, magnesium chloride and bovine serum albumin.

[0073] In this invention, the concentration of deoxyribonuclease is 100–720 U / mL, for example, it can be 100 U / mL, 200 U / mL, 300 U / mL, 400 U / mL, 500 U / mL, 600 U / mL, 700 U / mL, or 720 U / mL, etc.; the concentration of ribonuclease is 10–100 U / mL, for example, it can be 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, etc. The concentrations of the nuclease buffer solution are 50 mmol / L, 80 U / mL, 90 U / mL, or 100 U / mL, etc.; the concentration of the Tris-HCl buffer solution is 10 mmol / L; the concentration of the magnesium chloride is 10 mmol / L; and the concentration of the bovine serum albumin is 1 mg / mL. The pH of the nuclease buffer solution is 7–7.8. The nuclease treatment temperature is 37°C, and the time is 4–8 h, for example, 4 h, 5 h, 6 h, 7 h, or 8 h, etc., and the nuclease buffer solution is replaced with fresh solution every 2–4 h.

[0074] Step (5) of this invention involves washing and freeze-drying the nuclease-treated sample sequentially to obtain a whole intervertebral disc decellularized tissue engineering scaffold.

[0075] In this invention, the sample is washed continuously with PBS for 8 hours, then washed with distilled water, cleaned with anhydrous ethanol for 30 minutes, and then freeze-dried after natural drying.

[0076] In this invention, the freeze-drying conditions are: pre-cooling for 200 minutes, drying at -70°C for more than 24 hours, preferably 24 to 48 hours, and a vacuum degree of 0.1 Pa.

[0077] In some implementations, the sample needs to be washed with distilled water and then subjected to gradient dehydration with ethanol. The range of ethanol that can be used is (70%–99%). To achieve high-efficiency dehydration and reduce experimental errors, gradient dehydration with ethanol can be performed (gradient dehydration concentrations of 30%, 50%, 70%, and 99%).

[0078] The present invention also provides a whole intervertebral disc decellularized tissue engineering scaffold prepared by the above preparation method. The scaffold removes most of the cellular components and genetic material, while retaining a relatively complete extracellular matrix structure and mechanical properties, and can be used in the fields of intervertebral disc repair and regenerative medicine.

[0079] The present invention also provides the application of the above-mentioned whole intervertebral disc decellularized tissue engineering scaffold in the preparation of intervertebral disc repair implant materials.

[0080] The application of the whole intervertebral disc decellularized tissue engineering scaffold provided by this invention in the fields of tissue engineering and regenerative medicine.

[0081] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0082] In the embodiments of this invention, the PBS used is standard concentration PBS, not concentrated 1×PBS (0..01M).

[0083] Example 1

[0084] (1) Using a high-speed micro bone saw, the adjacent vertebrae were carefully cut off along the middle of the vertebral body of the fresh cattle tail purchased from the slaughterhouse, and finally a "disc-bone complex" sample was obtained, which was composed of the central intervertebral disc tissue and the bone of the vertebrae above and below it; the thickness of the vertebrae above and below the sample was 1-2 mm.

[0085] (2) Wash the sample with PBS for 1 hour at 4℃; smooth the surface of the cut upper and lower vertebrae with sandpaper; degrease the sample with a chloroform / methanol mixture at 4℃, ensuring full contact between the sample and the chloroform / methanol mixture; wash the sample three times with PBS after degreasing for 6 hours; then wash the sample with distilled water and anhydrous ethanol for 1 hour at 4℃.

[0086] (3) The defatted sample was frozen in liquid nitrogen at -196℃ for 10 min, and then placed in a water bath at 37℃ in 10 mMtris-HCl hypotonic buffer (pH 8.0) containing protease inhibitor for 30 min. The concentration of protease inhibitor was 1 mmol / L. After warming, the sample was washed with PBS containing protease inhibitor for 10 min (washing cycle 3 times) to obtain the pretreated sample.

[0087] (4) Wash the pretreated sample with distilled water first, and then dehydrate the sample in sequence with ethanol of concentration gradients of 30%, 50%, 70% and 99%. The treatment time for each concentration of ethanol should not be less than 30 min, and the total dehydration time should not be less than 2 h. After treatment, let it dry naturally for later use.

[0088] (5) Place the sample after the previous step into a Tris-HCl buffer containing Triton X-100 and PMSF (serine protease inhibitor). The concentration of Triton X-100 is 2%, the concentration of PMSF (serine protease inhibitor) is 1 mmol / L, the concentration of Tris-HCl buffer is 10 mmol / L, and the pH is 7.5. Then place it on a shaker at 4°C and shake continuously (frequency 180 r / min) for 4 to 8 hours. Rinse the sample with distilled water continuously for 3 hours.

[0089] (6) Place the sample after the previous step in a decellularization solution at 4°C. Specifically, the decellularization solution consists of a tris-HCl buffer (10mM tris-HCl, pH 8.0) containing 1% SDS, 0.2% EDTA and 10 KIU / mL aprotinin. The sample is treated in the decellularization solution for 24 hours, and ultrasonic elution is performed every 5 hours. Ultrasonic elution is performed by shaking on a shaker (frequency 80r / min) with an ultrasonic power of 12kHz for 1 hour.

[0090] (7) Wash the sample after the previous step with 0.5 mol / L potassium chloride and distilled water for 30 min, soak in anhydrous ethanol solution for 30 min, let it evaporate naturally in a ventilated place for 30 min, and then completely immerse the sample in tris-HCl buffer containing deoxyribonuclease, ribonuclease, magnesium chloride and bovine serum albumin at 37°C for 8 h (change the solution once every 4 hours). The concentration of deoxyribonuclease in the tris-HCl buffer is DNase 200 U / mL, RNase 10 U / mL, magnesium chloride 10 mmol / L, and bovine serum albumin 1 mg / mL; the concentration of the tris-HCl buffer is 50 mmol / L and the pH is 7.5.

[0091] (8) After washing the sample treated in the previous step with PBS for 8 hours (replace with fresh PBS solution every 2 hours), wash the sample with distilled water, clean with anhydrous ethanol, filter for 30 minutes, air dry, pre-cool for 200 minutes, dry at -70℃ for 48 hours with a vacuum degree of 0.1 Pa to obtain a decellularized sample scaffold.

[0092] Prepared scaffold structures that have not been treated with a freeze dryer, such as Figure 1 As shown, by Figure 1As can be seen, the stent conforms to the size of a human intervertebral disc prosthesis and maintains the anatomical shape of a natural intervertebral disc.

[0093] Example 2: Histological analysis (HE) of decellularized tissue engineering scaffolds

[0094] The scaffold prepared in Example 1 was cut into 1 mm thick samples and completely immersed in a sufficient amount of 4% paraformaldehyde (PFA) fixative. The samples were then placed in a 4°C refrigerator for 24 to 48 hours to ensure the fixative fully penetrated the porous structure of the scaffold and completely preserved its extracellular matrix morphology. After rinsing with running water overnight, the fixed samples were sequentially dehydrated by diluting them in 50%, 70%, 80%, 90%, and 95% ethanol for 2 hours each, and in 100% ethanol I and II for 1.5 hours each. They were then cleared by diluting them in xylene I and II for 1 hour each. Afterward, they were immersed in paraffin I and paraffin II for 3 hours each in a 60°C incubator. Finally, they were embedded using a paraffin embedding machine and sectioned continuously at a thickness of 5 μm using a microtome. The sections were then spread in 40°C warm water and transferred to glass slides, then baked at 60°C for 4 hours to ensure tight adhesion. The dried sections were dewaxed by immersing them sequentially in xylene I and II for 10 minutes each, followed by hydration in a gradient of 100%, 95%, 90%, 80%, and 70% ethanol for 5 minutes each, and then rinsed with running water for 5 minutes. Next, they were stained with Harris hematoxylin for 8 minutes, rinsed with running water for 1 minute, differentiated with 1% hydrochloric acid alcohol for 3 seconds, rinsed with running water for 15 minutes to regain blue color, counterstained with 0.5% eosin Y aqueous solution for 2 minutes, and rinsed briefly with running water for 5 seconds. Finally, they were dehydrated by a gradient of 70%, 80%, 90%, and 95% ethanol for 2 minutes each, thoroughly dehydrated in 100% ethanol I and II for 5 minutes each, cleared in xylene I and II for 5 minutes each, and then covered with a coverslip for mounting.

[0095] The prepared sections were placed under an optical microscope. First, the overall structure, pore distribution, integrity, and fiber arrangement of the extracellular matrix were observed in a panoramic view under a 4x objective lens. Finally, under a 40x objective lens, the field of view was carefully searched for any cell nuclei or nuclear fragments stained blue-purple (the sign of successful decellularization is the absence of any nuclear residue). The morphology, density, and retention of the matrix, such as collagen fibers and elastic fibers stained pink, were also evaluated in detail. At the same time, key fields of view were photographed and recorded.

[0096] HE-stained section structure as follows Figure 2 As shown, by Figure 2As can be seen, under 4x and 40x objectives on the bone tissue / annulus fibrosus / nucleus pulposus / endplate, the overall structure, pore distribution, integrity, and fibrous arrangement of the extracellular matrix of the scaffold were well preserved. Very few cell nuclei or nuclear fragments stained blue-purple were observed in the annulus fibrosus / nucleus pulposus / bone tissue, and the cartilaginous endplate also showed good elution. This material possesses the three-dimensional porous structure of a natural intervertebral disc, with a pore size ranging from 50-300 μm and a porosity greater than 90%.

[0097] Example 3: Biomechanical environment testing of cell tissue engineering scaffolds

[0098] The scaffold prepared using the freeze-drying process of Example 1 was placed in medical gauze soaked in phosphate-buffered saline (PBS) and incubated in a water bath at room temperature for 48 hours during the MTS test. Biomechanical tests were performed, including six zigzag cycles (the first 19 cycles were for pretreatment, and the last cycle was used for analysis) of compression at -200 N and 0.5 Hz, followed by a 10-minute creep test at -200 N and a maximum destructive stress test, all conducted using the MTS Criterion test system. Creep stress and strain were calculated based on the specimen's behavior during the creep process.

[0099] The support in the MTS test, such as Figure 3 As shown, both the experimental and control groups were able to achieve fatigue testing at 200N, and the compressive modulus of the freeze-dried stent was slightly higher than that of the fresh stent.

[0100] Example 4: Water Absorption Test of Cell Tissue Engineering Scaffold

[0101] 1. Equilibrate the lyophilized scaffold in a desiccator for 24 hours and record the initial mass (W0). 2. Immerse the scaffold in ddH2O, DMEM, and PBS (ensuring complete coverage of the sample) respectively, and place it on a shaker (37℃, 50rpm) to simulate a dynamic body fluid environment. Set time gradients (e.g., 5min, 15min, 30min, 1h, 2h, 4h, 24h, 48h), and take one set of samples at each time point (n=5). 3. After removing the samples, gently wipe away any free droplets on the surface with filter paper (avoid excessive squeezing). Weigh immediately (Wt), repeat 3 times, and take the average value. 4. When the water absorption rate changes by <5% over 3 consecutive time points, it is considered saturated water absorption.

[0102] The water absorption rate (Rw) is calculated based on the difference between the wet weight (Ww) and dry weight (Wd) of the sample. The formula is: Rw = (Ww - Wd) / Wd.

[0103] The water absorption test of the stent is as follows Figure 4As shown, the ddH2O (ultrapure water) group had the highest water absorption rate (143.1% ± 28.3%), significantly higher than the PBS group (116.8% ± 12.8%) and the DMEM group (101.5% ± 26.9%). Possible reasons: ① Difference in ionic strength: PBS and DMEM contain salt ions (such as Na+). + Cl - ① Osmotic pressure competition may inhibit water absorption by the scaffold; while ultrapure water, free from ion interference, can more easily penetrate into the scaffold. ② Compositional influence: DMEM contains amino acids, glucose, etc., which may partially adsorb into the scaffold pores, reducing free water binding sites.

[0104] The water absorption rate of the PBS group was between that of ddH2O and DMEM, which is consistent with the intermediate state of its physiological ion concentration.

[0105] The DMEM group had the lowest water absorption rate, which may be related to pore blockage or reduced surface energy caused by complex components (such as proteins and buffer systems).

[0106] Stent characteristic inference

[0107] The high water absorption capacity (all >100%) indicates that the scaffold has a porous hydrophilic structure, making it suitable as a cell carrier or sustained-release matrix.

[0108] Media-dependent characteristics suggest that in practical applications (such as in vivo implantation), the local microenvironment (such as tissue fluid composition) may significantly affect the scaffold hydration state, thereby influencing mechanical properties or cell behavior.

[0109] Example 5: Determination of DNA / GAG content in cell tissue engineering scaffolds

[0110] Take the scaffold prepared in Example 1 without freeze-drying, and extract approximately 25 mg of a homogeneous sample (nucleus pulposus / annulus fibrosus / cartilaginous endplate; accurate data for bone tissue was not obtained due to its hardness and technical limitations). Grind the sample in liquid nitrogen and place it in a centrifuge tube. Add 180 μL of Buffer GTL. Add 20 μL of Protein K, vortex to thoroughly mix the sample, and incubate at 56°C until the tissue is completely lysed. Add 200 μL of Buffer GL, vortex to mix thoroughly, and incubate at 70°C for 10 minutes. After brief centrifugation, add 200 μL of anhydrous ethanol and vortex to mix thoroughly. After brief centrifugation, add the entire solution to the adsorption column already loaded into the collection tube. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and return the adsorption column to the collection tube. Add 500 μL of Buffer GW1 to the adsorption column, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and return the adsorption column to the collection tube. Add 500 μL of Buffer GW2 to the adsorption column, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the adsorption tube. Centrifuge at 12000 rpm for 2 minutes, discard the waste liquid, and allow the adsorption column to air dry completely at room temperature for several minutes. Place the adsorption column in a new centrifuge tube, add 50-200 μL of Buffer GE or sterile water to the center of the column, incubate at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, and collect the DNA solution. Quantify the collected DNA solution by measuring its concentration at 280 nm using a microplate reader.

[0111] The same sample tissues were digested in papain buffer (125 mg / ml papain, 5 mM cysteine ​​hydrochloride and 5 mM disodium ethylenediaminetetraacetate dissolved in PBS) at 60°C for 12 hours. The resulting solution was quantified by measuring the absorbance of the supernatant at 525 nm using the DMMB colorimetric method.

[0112] DNA content assay results of the scaffold are as follows Figure 5 As shown, the DNA content in the experimental group was significantly reduced compared to the control group, falling below the decellularization standard of 50 ng / mg.

[0113] The results of GAG content determination of the stent are as follows: Figure 6 As shown in the figure, the experimental group had more GAG ​​content loss and was suitable for cell seeding.

[0114] Example 6 Histological analysis of cell tissue engineering scaffolds: COL1 / COL2 and Sirius staining

[0115] The scaffold prepared in Example 1 without freeze-drying was cut into 1 mm thick sample sections and stained with hematoxylin, eosin, and Picrosirius Red. Then, immunohistochemical analysis was performed on type I collagen (CoL1) and type II collagen (CoL2). A negative control group was included in all immunohistochemical samples. The specific steps are as follows:

[0116] Antigens were extracted from slides obtained from paraffin sections using sodium citrate heat induction. The slides were then treated with 3% hydrogen peroxide solution and incubated in the dark at room temperature for 25 min to block endogenous peroxidase. Buffer (PBS of 10% normal goat serum and 0.3% Triton X-100) was used for wetting and sealing. Next, the slides were incubated overnight at 4°C with primary antibodies (CoL1, CoL2). After washing three times with PBS (pH 7.40, 0.01M) for 5 min each time, the slides were incubated again with the corresponding secondary antibodies (HRP-conjugated goat anti-mouse, Proteintech, 1:100 dilution) in the dark at 37°C for 2 h. DAB development was then performed. The procedure was as follows: the slides were placed in PBS solution and washed three times on a shaker for 5 min each time. Next, the slides were slightly dried and DAB developing solution was added. The development time was controlled by microscopic observation; positive colors were brown and yellow. The sections were then rinsed with tap water to stop the color development, and then restained with hematoxylin. Finally, the sections were observed under a fluorescence microscope.

[0117] The results of COL1 / COL2 and Sirius staining sections of the scaffold are as follows: Figure 7 and 8 As shown, COL1 / COL2 content is relatively low after decellularization, making it suitable for cell inoculation.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a whole-intervertebral disc decellularized tissue engineering scaffold, characterized in that, Includes the following steps: (1) Take the intervertebral disc-bone complex of vertebrates as a sample, and perform surface polishing, degreasing and cryopreservation thawing treatment on the sample in sequence to obtain a pre-treated sample. (2) The pretreated sample was placed in Tris-HCl buffer containing surfactant and protease inhibitor and shaken. (3) Place the shaken sample in a decellularization solution for decellularization. (4) Place the decellularized sample in a nuclease buffer solution for nuclease treatment; (5) The nuclease-treated samples were washed and freeze-dried in sequence to obtain a whole intervertebral disc decellularized tissue engineering scaffold.

2. The preparation method according to claim 1, characterized in that, The intervertebral disc-bone complex is composed of central intervertebral disc tissue and the bone of the vertebrae above and below it; the thickness of the vertebral portions above and below the intervertebral disc-bone complex is 1 to 2 mm.

3. The preparation method according to claim 1, characterized in that, The degreasing treatment includes: immersing the polished sample in a mixed solution of chloroform and toluene; the temperature of the degreasing treatment is 4-30°C, and the time is 4-8 hours; the volume ratio of chloroform to toluene is 1-2:

1.

4. The preparation method according to claim 1, characterized in that, The cryopreservation and thawing process includes: freezing the defatted sample in liquid nitrogen and thawing it in a hypotonic buffer solution containing a protease inhibitor; the cryopreservation temperature is -196°C; the time is 3–15 min; the pH of the hypotonic buffer solution is 7.5–8; the concentration of the protease inhibitor in the hypotonic buffer solution is 1 mmol / L; and the thawing temperature is 37°C for 30 min.

5. The preparation method according to claim 1, characterized in that, The surfactant in step (2) includes Triton X-100, and the protease inhibitor includes serine protease inhibitor; the pH of the Tris-HCl buffer is 7.2-8; the concentration of the surfactant in the Tris-HCl buffer is 1-5%, and the concentration of the protease inhibitor is 1 mmol / L; the shaking speed is 160-240 r / min, the temperature is 4℃, and the time is 4-8 h.

6. The preparation method according to claim 1, characterized in that, The decellularization solution is a mixture of sodium dodecyl sulfate, ethylenediaminetetraacetic acid (EDTA), aprotinin, and Tris-HCl buffer; the concentration of sodium dodecyl sulfate in the decellularization solution is 0.1–1%, the concentration of EDTA is 0.1–0.5%, the concentration of aprotinin is 1–10 KIU / mL, and the concentration of Tris-HCl buffer is 10 mmol / L; the pH of the decellularization solution is 8. The decellularization process is carried out under shaking and ultrasound, with the shaking frequency being 80-120 r / min, the ultrasound power being 10-18 kHz, and the temperature being 4℃.

7. The preparation method according to claim 1, characterized in that, The nuclease buffer solution comprises deoxyribonuclease, ribonuclease, Tris-HCl buffer, magnesium chloride, and bovine serum albumin; the concentration of deoxyribonuclease in the nuclease buffer solution is 100–720 U / mL, the concentration of ribonuclease is 10–100 U / mL, the concentration of Tris-HCl buffer is 50 mmol / L, the concentration of magnesium chloride is 10 mmol / L, and the concentration of bovine serum albumin is 1 mg / mL; the pH value of the nuclease buffer solution is 7–7.8; the nuclease treatment temperature is 37°C, the time is 4–8 h, and the nuclease buffer solution is replaced with fresh solution every 2–4 h.

8. The preparation method according to claim 1, characterized in that, The freeze-drying temperature is -70℃, the time is 24 to 48 hours, and the vacuum degree is 0.1 Pa.

9. The whole intervertebral disc decellularized tissue engineering scaffold prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the whole intervertebral disc decellularized tissue engineering scaffold according to claim 9 in the preparation of intervertebral disc repair implant materials.

Images