Preparation method and application of nucleus pulposus acellular matrix hydrogel material

Abstract

The invention relates to the technical field of biological materials, in particular to a preparation method and application of a nucleus pulposus acellular matrix hydrogel material. The method comprises the following steps: taking a proper amount of lumbar nucleus pulposus tissues of pigs in a slaughter house, cleaning the lumbar nucleus pulposus tissues with PBS for several times, then taking part of the lumbar nucleus pulposus tissues for treatment, and subpackaging the residual nucleus pulposus tissues at-20 DEG C Preparing a decellularized liquid working solution; preparing an acellular nucleus pulposus matrix; cleaning the acellular nucleus pulposus matrix; evaluating acellular nucleus pulposus matrix genetic material residues and extracellular matrix preservation conditions; preparing a powdery nucleus pulposus acellular matrix; preparing a methacrylated acellular nucleus pulposus matrix; detecting the cross-linking performance of the methacrylated acellular nucleus pulposus matrix; through the mode, the efficient and complete nucleus pulposus acellular matrix can be obtained, and the nucleus pulposus acellular matrix hydrogel material which has good activity, proper mechanical property, quick crosslinking, injectability and strong applicability can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, and particularly relates to a preparation method and application of a nucleus pulposus acellular matrix hydrogel material. BACKGROUND

[0002] The acellular matrix material has good biocompatibility and low immunogenicity due to the retention of various core components in the natural tissue, and shows great application potential in the fields of tissue engineering and regenerative medicine, and provides a new idea for the repair and regeneration of nucleus pulposus tissue.

[0003] Currently, there are relevant studies on methods for modifying decellularized nucleus pulposus matrix. For example, Method 1 involves decellularizing the nucleus pulposus tissue without any modification and using it directly as a scaffold material. However, this method has limited application and a limited number of cells that can be loaded. Method 2 involves digesting the decellularized nucleus pulposus matrix powder in pepsin and then adding genipin to form a thermosensitive self-crosslinking hydrogel. However, the crosslinking of genipin depends on the number of amino groups, solution pH, temperature, and time, resulting in poor controllability and a slow crosslinking speed. Generally, it takes 48 hours to completely gel at 37°C, and low temperatures or low concentrations also contribute to the problem. Under certain conditions, the time required for cross-linking is significantly prolonged, and the color turns blue / dark blue during the cross-linking process, which is not conducive to observation under an optical microscope. Once the cross-linking is excessive, the material becomes hard and dense, which will greatly affect cell proliferation and survival. Method 3 is an injectable, photocross-linked decellularized matrix composite hydrogel and its preparation method and application (CN114712561A). In this method, the decellularized matrix powder is first mixed and stirred with an acidic pepsin solution to remove undigested matrix particles and obtain a decellularized matrix pregel solution. Then, the pregel solution is pre-cooled and an alkaline solution is added to adjust the temperature. The pH is adjusted, and finally, polyethylene glycol diacrylate and photoinitiator are dissolved in a pre-cooled buffer solution. After stirring evenly with the pre-gel solution at 0-8℃, photocrosslinking and curing are performed to obtain a decellularized matrix composite hydrogel. Although this preparation method is simple and easy to operate, the prepared composite hydrogel has high mechanical strength, long degradation time, and good biocompatibility. It can be used to load growth factors, inject them into the body, and then photocrosslink them to achieve sustainable release of growth factors to promote tissue repair and regeneration. However, the structure of polyethylene glycol diacrylate after crosslinking has high stability and low biodegradability. Method 4 (CN111184914A) discloses a method for constructing functionalized decellularized matrix biomaterials. By sequentially immersing decellularized animal tissues in methacrylic anhydride and propyl methacrylate-3-sulfonate solutions for specific functional modification, the tissues are then crosslinked with matrix materials using free radical polymerization to form an interpenetrating network structure to enhance their mechanical properties. However, this method has the limitation of relying on the original structure of animal tissues, and repeated exposure to organic solvent environments can easily lead to a decrease in biological activity, which limits the applicability and application of biological properties of the material. In addition, similar photocrosslinked decellularized matrix patents, such as a bladder decellularized matrix hydrogel, 3D bioprinted bladder patch and its preparation method and application (CN119838062A), involve freeze-drying the bladder decellularized matrix to obtain freeze-dried powder, adding hydrochloric acid pepsin solution for digestion, adding alkaline solution to adjust the pH, cooling and adding methacrylic anhydride to stir the reaction in the dark, and finally dialyzing with a dialysis membrane and freeze-drying to obtain methacrylamide bladder decellularized matrix.

[0004] In summary, existing modification methods either have limited application scope and insufficient cell loading, or poor controllability of the cross-linking process, slow cross-linking speed and impact on cell proliferation and survival, or unsatisfactory biodegradability of the cross-linked structure, or rely on the original tissue structure and are prone to decline in biological activity. As a result, it is difficult to obtain decellularized nucleus pulposus matrix hydrogel materials with good activity, suitable mechanical properties, rapid cross-linking, injectability and strong applicability. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying a decellularized nucleus pulposus matrix hydrogel material, aiming to solve the technical problem in the prior art of obtaining a decellularized nucleus pulposus matrix hydrogel material with good activity, suitable mechanical properties, rapid cross-linking, injectability, and strong applicability.

[0006] To achieve the above objectives, the present invention provides a method for preparing a decellularized nucleus pulposus matrix hydrogel material, comprising the following steps: Take an appropriate amount of lumbar nucleus pulposus tissue from pigs at the slaughterhouse, wash it several times with PBS, process a portion of it, and store the remaining nucleus pulposus tissue at -20°C for later use. Prepare the decellularization working solution; Preparation of decellularized nucleus pulposus matrix; Cleanse the decellularized nucleus pulposus matrix; Assessment of residual genetic material in the decellularized nucleus pulposus matrix and preservation of the extracellular matrix; Preparation of powdered decellularized nucleus pulposus matrix; Preparation of methacrylated decellularized nucleoplastin; The cross-linking properties of methacrylamide-derived decellularized nucleus pulposus matrix were tested.

[0007] In the step of preparing the decellularized working solution: The detergent solution consisted of 10 mM Tris-HCl buffer, 3% Triton X-100, 0.1% EDTA, 0.2 mg / mL LRnase, 0.2 mg / mL DNase, and 10 KIU / mL protein inhibitor.

[0008] In the step of preparing the decellularized nucleus pulposus matrix: The nucleus pulposus tissue washed with PBS was placed in pre-cooled double-distilled water at 4°C; Repeated freeze-thaw cycles at -80°C and 37°C in a water bath for 3 days; Remove the supernatant by centrifugation, 500g, centrifuge for 5min, and retain the lower layer of jelly-like nucleus pulposus tissue; The nucleus pulposus tissue was removed and placed in the chamber of a circulating perfusion hydrostatic bioreactor; The chamber and peristaltic pump were filled with decellularized solution. The washing solution was refreshed every 2 hours by the peristaltic pump at a flow rate of 10 ml / min, and the process was continued for 24 hours.

[0009] In the step of washing the decellularized nucleus pulposus matrix: Decellularized tissue was removed from the bioreactor and washed continuously with sterile PBS containing 10 KIU / ml protein inhibitors at 4°C for 24 hours, with the PBS being replaced every 4 hours. Remove residual reagents and remove the supernatant by centrifugation at 500g for 5 minutes, retaining the decellularized nucleus pulposus matrix for later use.

[0010] Among the steps involved in assessing the residual genetic material in the decellularized nucleus pulposus matrix and the preservation of the extracellular matrix: Select appropriate amounts of fresh nucleus pulposus and decellularized nucleus pulposus matrix, and compare the preservation status of type II collagen and ACAN by ELISA. DNA fluorescence staining and DAPI staining are used to assess the residual DNA content. Histological staining was performed, and the preservation of the matrix was assessed by H&E staining and alcine blue staining.

[0011] In the step of preparing powdered decellularized nucleus pulposus matrix: Freeze-dry the decellularized nucleus pulposus matrix; The decellularized nucleus pulposus matrix was freeze-dried and ground into powder. The powder was then resuspended in 10 mM hydrochloric acid and 0.2% porcine trypsin and reacted at room temperature for 1 hour until the solution became clear. The reaction was terminated by adding 10 mM Tris-HCl buffer containing 10 mM sodium hydroxide to obtain a decellularized matrix solution. After dialysis with deionized water for 3 days to remove excess inorganic salts, the remaining solution was freeze-dried into decellularized nucleus pulposus matrix powder for later use.

[0012] In the step of preparing methacrylated decellularized nucleoplastin matrix: Dissolve 0.5g of decellularized nucleus pulposus matrix powder in 25ml of deionized water, put it into a brown wide-mouth bottle, place it on an ice bath, and slowly add 0.5mL of methacrylic anhydride to the supernatant using a micro-injection pump; After the methacrylic anhydride has been added, add 0.5 mL of 5M NaOH to the system and allow it to react in the dark for 24 hours. After the reaction is complete, weigh 2000g and centrifuge for 10 minutes. After collecting the supernatant, transfer all the liquid to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialyze in a 37°C water bath for 3 days, replacing the dialysis solution in the water bath with pure water every 4 hours. After dialysis is completed, freeze at -80 ℃ for freeze-drying and use.

[0013] In the step of detecting the crosslinking properties of methacrylamide-treated decellularized nucleus pulposus matrix: Take an appropriate amount of methacrylated decellularized nucleus pulposus matrix and dissolve it in a solution containing 0.5% lithium phenyl phosphate to prepare a 10% stock solution of methacrylated decellularized nucleus pulposus matrix; 1000 μL of the mother liquor was diluted to prepare 4%, 6%, and 8% methacrylamide decellularized nucleus pulposus matrix, respectively. The crosslinking properties and mechanical properties of different concentrations were verified by irradiation with 485 nm blue light. The microstructure of the fully cross-linked 8% methacrylamide decellularized nucleus pulposus matrix was observed using scanning electron microscopy.

[0014] The present invention also provides one.

[0015] The present invention also provides the application of the preparation method of the decellularized nucleus pulposus matrix hydrogel material in promoting tissue repair and regeneration in vivo.

[0016] This invention discloses a method for preparing and applying a decellularized nucleus pulposus matrix hydrogel material. First, a suitable amount of lumbar nucleus pulposus tissue from pigs is collected at a slaughterhouse. After washing several times with PBS, a portion is processed, and the remaining nucleus pulposus tissue is aliquoted at -20°C and stored for later use. Then, a decellularization working solution is prepared, consisting of a detergent solution composed of 10 mM Tris-HCl buffer, 3% Triton X-100, 0.1% EDTA, 0.2 mg / mL LRnase, 0.2 mg / mL DNase, and 10 KIU / mL protein inhibitor. Next, a decellularized nucleus pulposus matrix is ​​prepared, washed, and the genetic material residue and extracellular matrix preservation of the decellularized nucleus pulposus matrix are evaluated. Then, powdered decellularized nucleus pulposus matrix is ​​prepared, followed by the preparation of methacrylamide-modified decellularized nucleus pulposus matrix. Finally, the cross-linking properties of the methacrylamide-modified decellularized nucleus pulposus matrix are tested. Through this method, the decellularized matrix material retains the core ECM components of natural tissue, such as collagen, proteoglycans, GAGs, elastin, and adhesion molecules. Compared to other materials, the composition exhibits excellent biocompatibility and low immunogenicity. Addressing the shortcomings of existing applications in the fabrication of decellularized nucleus pulposus matrix materials, this invention utilizes our self-developed hydrostatic circulating perfusion bioreactor to obtain a highly efficient and complete decellularized nucleus pulposus matrix. Furthermore, through methacrylation modification, a highly bioactive and injectable, rapidly cross-linked photocrosslinked decellularized nucleus pulposus matrix is ​​prepared, exhibiting strong mechanical properties and significantly improving the applicability of the decellularized nucleus pulposus matrix material. This invention enables the acquisition of a highly efficient and complete decellularized nucleus pulposus matrix, achieving a decellularized nucleus pulposus matrix hydrogel material with good activity, suitable mechanical properties, rapid cross-linking, injectability, and strong applicability. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart illustrating the steps of the preparation method of the decellularized nucleus pulposus matrix hydrogel material of the present invention.

[0019] Figure 2 This is a flowchart of steps S300 of the present invention.

[0020] Figure 3 This is a flowchart of steps S400 of the present invention.

[0021] Figure 4 This is a flowchart of steps S500 of the present invention.

[0022] Figure 5 This is a flowchart of steps S600 of the present invention.

[0023] Figure 6 This is a flowchart of steps S700 of the present invention.

[0024] Figure 7 This is a flowchart of steps S800 of the present invention.

[0025] Figure 8 This is a flowchart illustrating the preparation process of the decellularized nucleus pulposus matrix hydrogel material of the present invention.

[0026] Figure 9 This is a graph showing the crosslinking performance of the decellularized nucleus pulposus matrix hydrogel material of the present invention.

[0027] Figure 10 This is a graph showing the physical performance evaluation and comparison of the decellularized matrix hydrogel material of the nucleus pulposus of the present invention.

[0028] Figure 11 This is a biocompatibility evaluation diagram of the decellularized matrix hydrogel material of the nucleus pulposus of the present invention. Detailed Implementation

[0029] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application.

[0030] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0031] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0032] Please see Figures 1-11 This invention provides a method for preparing a decellularized nucleus pulposus matrix hydrogel material, comprising the following steps: S100: Take an appropriate amount of lumbar nucleus pulposus tissue from pigs at the slaughterhouse, wash it several times with PBS, process a portion of it, and store the remaining nucleus pulposus tissue at -20°C for later use. S200: Prepare the decellularization working solution by combining 10 mM Tris-HCl buffer, 3% Triton X-100, 0.1% EDTA, 0.2 mg / mL LRnase, 0.2 mg / mL DNase, and 10 KIU / mL protein inhibitor to form a washing solution; S300: Preparation of decellularized nucleus pulposus matrix.

[0033] In this embodiment, firstly, an appropriate amount of lumbar nucleus pulposus tissue from pigs is taken from the slaughterhouse, washed several times with PBS, and a portion is processed. The remaining nucleus pulposus tissue is aliquoted at -20°C and stored for later use. Next, a decellularized nucleus pulposus working solution is prepared, consisting of 10 mM Tris-HCl buffer, 3% Triton X-100, 0.1% EDTA, 0.2 mg / mL LNase, 0.2 mg / mL DNase, and 10 KIU / mL protein inhibitor. Then, decellularized nucleus pulposus matrix (DNPM) is prepared. The specific process for preparing decellularized nucleus pulposus matrix (DNPM) is as follows: S301: Place the nucleus pulposus tissue washed with PBS in pre-cooled double-distilled water at 4°C; S302: Repeated freeze-thaw cycles of -80℃ freezer and 37℃ water bath for 3 times; S303: Remove the supernatant by centrifugation, 500g, centrifuge for 5min, and retain the lower layer of jelly-like nucleus pulposus tissue; S304: Remove the nucleus pulposus tissue and place it in the chamber of the circulating perfusion hydrostatic bioreactor; S305: The chamber and peristaltic pump are filled with decellularized solution. The washing solution is refreshed every 2 hours by the peristaltic pump at a flow rate of 10 ml / min, and the system operates continuously for 24 hours.

[0034] In the above process, the nucleus pulposus tissue washed with PBS was first placed in pre-cooled double-distilled water at 4°C, and then subjected to three cycles of freeze-thaw in a -80°C freezer and a 37°C water bath. The supernatant was then removed by centrifugation (500g, centrifuged for 5 min), leaving the lower layer of jelly-like nucleus pulposus tissue. The nucleus pulposus tissue was then removed and placed in the chamber of a circulating perfusion hydrostatic bioreactor. Subsequently, the chamber and peristaltic pump were filled with decellularized solution. The washing solution was refreshed every 2 hours by the peristaltic pump at a flow rate of 10 ml / min, and the process was continued for 24 hours.

[0035] S400: Cleanses decellularized nucleus pulposus matrix.

[0036] In this embodiment, the specific process of washing the decellularized nucleus pulposus matrix is ​​as follows: S401: Remove decellularized tissue from the bioreactor and wash it continuously with sterile PBS containing 10 KIU / ml protein inhibitors at 4°C for 24 hours, changing the PBS every 4 hours. S402: Remove residual reagents and remove the supernatant by centrifugation. Centrifuge at 500g for 5 minutes and retain the decellularized nucleus pulposus matrix for later use.

[0037] In the above process, decellularized tissue was taken out from the bioreactor and washed continuously with sterile PBS containing 10 KIU / ml protein inhibitor at 4°C for 24 hours. The PBS was replaced every 4 hours. Then, residual reagents were removed, and the supernatant was removed by centrifugation at 500g for 5 minutes. The decellularized nucleus pulposus matrix (DNPM) was retained for later use.

[0038] S500: Assessment of residual genetic material in decellularized nucleus pulposus matrix and preservation of extracellular matrix.

[0039] In this embodiment, the assessment of residual genetic material in the decellularized nucleus pulposus matrix and the preservation of the extracellular matrix is ​​carried out as follows: S501: Select an appropriate amount of fresh nucleus pulposus and decellularized nucleus pulposus matrix, and compare the preservation status of type II collagen and ACAN by ELISA. DNA fluorescence staining and DAPI staining are used to assess the residual DNA content. S502: Perform histological staining, and assess the preservation of the matrix by H&E staining and alcine blue staining.

[0040] In the above process, an appropriate amount of fresh nucleus pulposus and decellularized nucleus pulposus matrix (DNPM) were selected. The preservation status of type II collagen and ACAN was detected and compared by ELISA. The residual DNA content was assessed by DNA fluorescence staining and DAPI staining. Then, histological staining was performed, and the matrix preservation status was assessed by H&E staining and alcine blue staining.

[0041] S600: Prepare powdered decellularized nucleus pulposus matrix.

[0042] In this embodiment, the powdered decellularized nucleus pulposus matrix is ​​prepared through the following process: S601: Freeze-drying of decellularized nucleus pulposus matrix; S602: After freeze-drying the decellularized nucleus pulposus matrix, grind it into powder, and resuspend the powder in 10mM hydrochloric acid and 0.2% porcine trypsin. React at room temperature for 1 hour until the solution becomes clear. S603: The reaction was terminated by adding 10mM Tris-HCl buffer containing 10mM sodium hydroxide to obtain a decellularized matrix solution; S604: Dialyze with deionized water for 3 days to remove excess inorganic salts, then freeze-dry the remaining solution into decellularized nucleus pulposus matrix powder for later use.

[0043] In the above process, the decellularized nucleus pulposus matrix (DNPM) was first freeze-dried, then the freeze-dried DNPM was ground into powder, and the powder was resuspended in 10 mM hydrochloric acid and 0.2% porcine trypsin. The reaction was carried out at room temperature for 1 hour until the solution became clear. Then, the reaction was terminated by adding 10 mM Tris-HCl buffer containing 10 mM sodium hydroxide to obtain the decellularized matrix solution. Subsequently, the solution was dialyzed with deionized water for 3 days to remove excess inorganic salts. The remaining solution was then freeze-dried into decellularized nucleus pulposus matrix (DNPM) dry powder for later use.

[0044] S700: Preparation of methacrylamide-derived decellularized nucleus pulposus matrix.

[0045] In this embodiment, the preparation of methacrylamide-derived decellularized nucleoplastin matrix is ​​carried out through the following process: S701: Dissolve 0.5g of decellularized nucleus pulposus matrix powder in 25ml of deionized water, put it into a brown wide-mouth bottle, place it on an ice bath, and slowly add 0.5mL of methacrylic anhydride to the supernatant using a micro-injection pump; S702: After the methacrylic anhydride has been added, add 0.5 mL of 5M NaOH to the system and react the system in the dark for 24 h. S703: After the reaction is complete, weigh 2000g and centrifuge for 10 minutes. After taking the supernatant, transfer all the liquid to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialyze in a 37-degree water bath for 3 days, replacing the dialysis solution in the water bath with pure water every 4 hours. S704: After dialysis is completed, freeze at -80 ℃ and freeze-dry for later use.

[0046] In the above process, 0.5 g of decellularized nucleus pulposus matrix (DNPM) powder was first dissolved in 25 ml of deionized water and placed in a brown wide-mouth bottle. The bottle was placed on an ice bath, and 0.5 mL of methacrylic anhydride was slowly added dropwise to the supernatant using a micro-injection pump. After the methacrylic anhydride was completely added, 0.5 mL of 5M NaOH was added dropwise to the system, and the system was kept in the dark for 24 h. After the reaction was completed, 2000 g was weighed and centrifuged for 10 min. The supernatant was collected, and all the liquid was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed in a 37°C water bath for 2 days, with the dialysis solution in the water bath replaced with pure water every 4 h. After the dialysis was completed, the solution was frozen at -80°C and freeze-dried for later use.

[0047] S800: Detects the crosslinking properties of methacryloylated decellularized nucleus pulposus matrix.

[0048] In this embodiment, the crosslinking properties of methacrylamide-treated decellularized nucleus pulposus matrix were tested, and the specific process was as follows: S801: Dissolve an appropriate amount of methacrylated decellularized nucleus pulposus matrix in a solution containing 0.5% lithium phenyl phosphate to prepare a 10% stock solution of methacrylated decellularized nucleus pulposus matrix; S802: Take 1000ul of the mother liquor and dilute it to prepare 4%, 6% and 8% methacrylamide decellularized nucleus pulposus matrix respectively. After irradiation with 485nm blue light, verify the crosslinking performance and mechanical properties of different concentrations. S803: The microstructure of the fully cross-linked 8% methacrylamide decellularized nucleus pulposus matrix was observed using scanning electron microscopy.

[0049] In the above process, an appropriate amount of methacrylated decellularized nucleus pulposus matrix (DNPM-MA) was first dissolved in a solution containing 0.5% lithium phenyl phosphate to prepare a 10% methacrylated decellularized nucleus pulposus matrix (DNPM-MA) stock solution. Then, 1000 μL of the stock solution was diluted to 4%, 6%, and 8% methacrylated decellularized nucleus pulposus matrix (DNPM-MA) respectively. The crosslinking performance and mechanical properties of different concentrations were verified by irradiation with 485 nm blue light. Subsequently, the microstructure of the fully crosslinked 8% methacrylated decellularized nucleus pulposus matrix (DNPM-MA) was observed by scanning electron microscopy.

[0050] Furthermore, see Figure 8Powdered DNPM was prepared by decellularization and freeze-drying. The modified Freytes method

[27] was used to gel the DNPM powder. This method combines enzymatic digestion and homogenization processes to methacrylate DNPM and synthesize photocrosslinkable DNPM-MA hydrogels. This reaction is similar to that of GelMA

[28] , HA-MA

[21] and other decellularized cartilage methacrylate modified hydrogels [29, 30]. In this study, porcine nucleus pulposus tissue was used as raw material to prepare DNPM gel, which was then used to prepare DNPM-MA hydrogel.

[0051] Furthermore, see Figure 9 We used a combination of chemical (Triton method) and biological (deoxyribonuclease and ribonuclease) methods for decellularization, and the specific procedure is described in our previous study

[20] . HE staining, DAPI staining, and quantitative detection of DNA content in fresh and decellularized nucleus pulposus samples showed that this method can effectively achieve decellularization ( Figure 9 BC and Figure 9 FG). Alcian blue staining, Picrosirius Red staining, and quantitative ELISA were used to detect glycosaminoglycans (GAG) and type II collagen (ColII), two key components of the nucleus pulposus matrix. The results showed that the contents of GAG and ColII in decellularized samples were slightly lower than those in fresh samples. Figure 9 DE and Figure 9 HI).

[0052] Furthermore, see Figure 10 The synthesis of DNPM-MA is based on the methacrylation reaction ( Figure 10 A), its ^1H-NMR spectrum showed a clear signal peak in the 5.2-5.8 ppm range, indicating that the methacrylate group had been successfully grafted onto the amino group of DNPM ( Figure 10 B). Given that the nucleus pulposus matrix is ​​mainly composed of type II collagen, we prepared Col-MA hydrogels based on porcine ColII via methacrylate esterification

[31] as a control group. Under the condition of irradiation with only 365 nm wavelength ultraviolet light for 30 seconds, both Col-MA and DNPM-MA precursor solutions could rapidly crosslink to form a gel structure ( Figure 10 C1-3). We then cast it into a hexagonal star mold to evaluate its shape retention. Except for 5% DNPM-MA, the 5% Col-MA, 8% and 10% DNPM-MA hydrogels all exhibited high shape fidelity after demolding. Figure 10 D, Figure S1). (As shown in Figure S1) Figure 10As shown in Figure E, scanning electron microscopy (SEM) images of the crosslinked hydrogels reveal that the pore size gradually decreases with increasing DNPM-MA concentration. Notably, the 5% Col-MA and 10% DNPM-MA hydrogels exhibit a uniform porous microstructure with comparable pore size and porosity. Figure 10 E, Figure S1). Mechanical analysis shows that the compressive modulus of all DNPM-MA hydrogels is lower than that of Col-MA (E, Figure S1). Figure 10 FG). However, with the increase of DNPM-MA concentration, its mechanical properties are significantly enhanced. Among them, the compressive modulus of 10% DNPM-MA hydrogel has reached the mechanical property requirements for constructing tissue-engineered nucleus pulposus (TE-NP). Figure 10 This conclusion is consistent with previous studies [5, 19]. Furthermore, the swelling capacity of DNPM-MA hydrogels is generally higher than that of Col-MA, and gradually decreases with increasing DNPM-MA concentration (FG). Figure 10 HI). The swelling properties of both 5% Col-MA and 10% DNPM-MA hydrogels are suitable for use in injectable TE-NP constructs. They maintain an appropriate volume after crosslinking, avoiding the risk of excessive expansion of the intervertebral disc cavity. Figure 10 HI).

[0053] The present invention also provides the application of the preparation method of the decellularized nucleus pulposus matrix hydrogel material in promoting tissue repair and regeneration in vivo.

[0054] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein.

[0055] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method of preparing a nucleus pulposus acellular matrix hydrogel material, characterized by, Comprising the following steps: At the slaughterhouse, take the pig's lumbar nucleus pulposus tissue, wash it several times with PBS, and then process part of it. The remaining nucleus pulposus tissue is stored at -20°C for future use. Prepare the decellularization solution working solution. Prepare the decellularized nucleus pulposus matrix. Wash the decellularized nucleus pulposus matrix. Evaluate the residual genetic material and extracellular matrix preservation of the decellularized nucleus pulposus matrix. Prepare the powdered nucleus pulposus decellularized matrix. Prepare the methacrylated decellularized nucleus pulposus matrix. Test the cross-linking properties of the methacrylated decellularized nucleus pulposus matrix.

2. The method of claim 1, wherein the preparation of the nucleus pulposus acellular matrix hydrogel material is characterized by, In the step of preparing the decellularization solution working solution: Prepare a detergent solution composed of 10 mM Tris-HCl buffer, 3% Triton X-100, 0.1% EDTA, 0.2 mg / mL Rnase, 0.2 mg / mL DNase, and 10 KIU / mL protease inhibitor.

3. The method of claim 2, wherein the nucleus pulposus acellular matrix hydrogel material is prepared by the steps of: In the step of preparing the decellularized nucleus pulposus matrix: Place the PBS-washed nucleus pulposus tissue in pre-cooled double distilled water at 4°C. Repeat freezing and thawing in a -80°C freezer and a 37°C water bath for 3 cycles. Remove the supernatant by centrifugation at 500g for 5 minutes, and retain the lower jellied nucleus pulposus tissue. Remove the nucleus pulposus tissue and place it in the chamber of a circulating perfusion hydrostatic bioreactor. Fill the chamber with the decellularization solution and use a peristaltic pump to update the washing solution every 2 hours at a flow rate of 10 ml / min. Continue working for 24 hours.

4. The method for preparing the decellularized nucleus pulposus matrix hydrogel material as described in claim 3, characterized in that, In the step of washing the decellularized nucleus pulposus matrix: Remove the decellularized tissue from the bioreactor and continuously shake it in sterile PBS containing 10 KIU / mL protease inhibitor at 4°C for 24 hours. Replace the PBS every 4 hours. Remove the residual reagents and remove the supernatant by centrifugation at 500g for 5 minutes. Retain the decellularized nucleus pulposus matrix for future use.

5. The method for preparing the decellularized nucleus pulposus matrix hydrogel material as described in claim 4, characterized in that, In the step of evaluating the residual genetic material and extracellular matrix preservation of the decellularized nucleus pulposus matrix: Select an appropriate amount of fresh nucleus pulposus and decellularized nucleus pulposus matrix. Use ELISA to detect and compare the preservation of type II collagen and ACAN in both. Use DNA fluorescent staining and DAPI staining to evaluate the residual DNA content. Perform histological staining and evaluate the matrix preservation by H&E staining and Alcian blue staining.

6. The method of claim 5, wherein the nucleus pulposus acellular matrix hydrogel material is prepared by the steps of: In the step of preparing the powdered nucleus pulposus decellularized matrix: Freeze-dry the decellularized nucleus pulposus matrix. Grind the freeze-dried decellularized nucleus pulposus matrix into powder and resuspend it in 10 mM hydrochloric acid and 0.2% porcine trypsin. React at room temperature for 1 hour until the solution is transparent. Add 10 mM Tris-HCl buffer containing 10 mM sodium hydroxide to terminate the reaction and obtain the decellularized matrix solution. Use deionized water for dialysis for 3 days. After removing excess inorganic salts, freeze-dry the remaining solution into a dry powder of nucleus pulposus decellularized matrix for future use.

7. The method for preparing the decellularized nucleus pulposus matrix hydrogel material as described in claim 6, characterized in that, In the step of preparing the methacrylated decellularized nucleus pulposus matrix: Dissolve 0.5 g of the dry powder of nucleus pulposus decellularized matrix in 25 ml of deionized water, place it in a brown wide-mouth bottle, and place it in an ice bath. Slowly add 0.5 mL of methacrylic anhydride to the supernatant using a microsyringe pump. After the addition of methacrylic anhydride is complete, add 0.5 mL of 5M NaOH to the system and react in the dark for 24 hours. After the reaction is completed, 2000g, centrifugal 10min, take supernatant after all liquid transfer to the molecular weight of 3500Da dialysis bag, 37 degree water bath in dialysis liquid, every 4h with pure water change dialysis liquid in water bath; Wait for dialysis, -80℃ freeze, freeze drying for standby.

8. The method for preparing the decellularized nucleus pulposus matrix hydrogel material as described in claim 7, characterized in that, In the step of detecting the crosslinking performance of the methacrylated decellularized nucleus pulposus matrix: Take an appropriate amount of methacrylated decellularized nucleus pulposus matrix and dissolve it with a lithium phenylphosphate solution containing 5 parts per thousand to prepare a 10% methacrylated decellularized nucleus pulposus matrix mother liquor; Each take 1000ul mother liquor respectively dilute into 4%, 6%, 8% methacrylated decellularized nucleus pulposus matrix, verify the crosslinking performance and mechanical properties of different concentrations after 485nm blue light irradiation; Use scanning electron microscope to observe the microstructure of the completely crosslinked 8% methacrylated decellularized nucleus pulposus matrix.

9. The use of the preparation method of the nucleus pulposus decellularized matrix hydrogel material according to claim 8 to promote tissue repair and regeneration in vivo.

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