Fat acellular matrix hydrogel material and preparation method thereof

Abstract

The invention provides a fat acellular matrix hydrogel material and a preparation method thereof. The fat freeze-dried powder is subjected to decellularization, digestion, acid-base balance regulation and gelation to prepare the fat freeze-dried powder. According to the method disclosed by the invention, a chemical decellularization method that liquid nitrogen freeze crushing is combined with 0.5% SDS and 1% Triton X-100 compound decellularization liquid is adopted, and fat is further removed through isopropyl alcohol treatment; adipose tissues can be uniformly crushed into powder through liquid nitrogen freezing and crushing, the action area of decellularization liquid is increased, the thoroughness of decellularization is remarkably improved, and the problem of fat or decellularization liquid residue can be avoided through multiple times of pure water and alcohol replacement treatment; according to the method, the mild decellularization solution is adopted for treatment for 12 hours, so that damage to key components of the matrix due to high-concentration chemical reagents and long-time action is effectively avoided. According to the method disclosed by the invention, immunological rejection caused by cell residues and adverse effects of fat residues on biocompatibility and mechanical properties can be remarkably reduced, so that the clinical applicability of the hydrogel is improved.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a fat decellularized matrix hydrogel material and a preparation method thereof. Background Art

[0002] In recent years, adipose decellularized extracellular matrix (adECM) has attracted much attention in the field of tissue repair and regenerative medicine. Adipose tissue has become an ideal material for soft tissue filling and defect repair because of its wide source, easy access, and rich extracellular matrix components (such as collagen, fibronectin, glycosaminoglycans, etc.). By treating adipose tissue with decellularization technology, it is possible to retain the active components of the matrix while removing cellular components and antigenic substances, and provide a three-dimensional microenvironment for cell migration, proliferation and differentiation. In order to facilitate operation and adapt to clinical needs, adECM is further prepared into a hydrogel form, which can significantly improve its plasticity and tissue compatibility. At present, the preparation method of adECM hydrogel usually includes decellularization of adipose tissue, formation and dissolution of matrix powder, gelation steps, etc. However, different decellularization processes and hydrogel preparation methods have a significant impact on the bioactivity, mechanical properties and clinical applicability of the material. In practical applications, how to achieve efficient decellularization, maximize the retention of matrix activity and improve the comprehensive performance of hydrogels is still an important technical problem in this field.

[0003] Although a variety of solutions have been proposed for the preparation of adECM hydrogel materials, the existing technology still has the following defects and shortcomings:

[0004] (1) Incomplete decellularization and fat residue issues

[0005] Among the existing traditional decellularization methods, physical methods (such as freeze-thaw method) and chemical methods (such as single use of SDS, Triton X-100, etc.) often cannot completely remove the cellular components and fat in adipose tissue. Cell residues may trigger immune rejection reactions, while fat residues affect the biocompatibility, mechanical properties and material stability of hydrogels. Existing technologies are difficult to effectively solve the problems of incomplete decellularization and fat residues, thus limiting their clinical applications.

[0006] (2) Matrix structure destruction and loss of biological activity

[0007] Although chemical decellularization is highly efficient, it can easily damage key components of the matrix, such as collagen and glycosaminoglycans. Especially under high concentrations of SDS (sodium dodecyl sulfate solution) or long-term treatment, the biological activity of the matrix may be significantly reduced, weakening its gelation ability and its support for tissue repair.

[0008] (3) Hydrogel performance needs to be optimized

[0009] The adECM hydrogels currently prepared still have deficiencies in terms of gelation time and stability. Long gelation time will affect its operational convenience and limit its practical application in tissue filling and repair. And unstable performance may make it difficult to provide sufficient support in tissue repair.

[0010] Based on the above-mentioned technical deficiencies, a new method for preparing adipose decellularized matrix hydrogel material is urgently needed. Summary of the invention

[0011] The purpose of the present invention is to provide a fat decellularized matrix hydrogel material and a preparation method; the present invention aims to solve the problems of incomplete decellularization, fat residue, matrix structure destruction, and poor hydrogel performance in the prior art. The present invention adopts a more efficient and gentle decellularization and degreasing process, which can effectively remove the cells and fat components in the adipose tissue, while retaining its natural extracellular matrix structure, ensuring the bioactivity and stability of the hydrogel, and providing a more effective and applicable fat decellularized matrix hydrogel material for clinical application.

[0012] The present invention is achieved through the following technical solutions:

[0013] The invention relates to a fat decellularized matrix hydrogel material, which is prepared from freeze-dried fat powder after decellularization, digestion, acid-base balance adjustment and gelation.

[0014] The present invention also relates to a method for preparing the aforementioned adipose decellularized matrix hydrogel material, comprising the following steps:

[0015] Step 1, preparation of decellularized matrix powder

[0016] (1.1) Wash and chop the adipose tissue, add pure water to cover the tissue, and freeze at -80℃ overnight;

[0017] (1.2) Move the frozen adipose tissue to a freeze dryer and freeze dry for 48 hours until the tissue is completely dry;

[0018] (1.3) Grinding into powder: The freeze-dried tissue is ground into fine powder using a grinding dish in liquid nitrogen and stored at -20°C after processing. Using liquid nitrogen freezing and crushing can evenly break the fat tissue into powder, increase the effective area of ​​the decellularization solution, and significantly improve the thoroughness of decellularization.

[0019] (1.4) Decellularization:

[0020] (a) Preparation of decellularization solution: 0.5% SDS and 1% TritonX-100 solution were mixed;

[0021] (b) Decellularization: 2.5 ml of decellularization solution was added to each milliliter of lyophilized fat powder, and the mixture was shaken at 37°C for 12 hours at a speed of 250 rpm. The shaking time was 12 hours, which greatly reduced the damage to matrix components such as collagen and glycosaminoglycans, and retained the key biological activity and three-dimensional microstructure of adECM.

[0022] (c) Cleaning the decellularization solution: After decellularization, rinse with pure water several times until the sample is clean;

[0023] (1.5) Degreasing treatment: The sample was immersed in isopropanol and shaken at 37°C for 24 h. It was then washed twice with pure water, treated with 75% alcohol for 30 min, and finally rinsed three times with pure water. The pure water and alcohol treatment were replaced several times during the washing process to effectively avoid the problem of fat or decellularization fluid residue.

[0024] (1.6) The cleaned samples were stored at -80°C overnight and freeze-dried for 48 hours. The powder was white and translucent and was stored at -20°C.

[0025] Step 2, Decellularized Matrix Hydrogel Preparation

[0026] (2.1) Digestion of matrix powder: Add 0.1 M HCl solution and pepsin to the adipose decellularized matrix powder and stir for 48 h until no visible particles are left. Pepsin digestion can efficiently dissolve adECM powder while maintaining the activity of its key components.

[0027] (2.2) Adjusting the acid-base / ion balance: After adjusting the solution pH to 7.3-7.4 using NaOH, 10X PBS was added at a total volume of 10% to restore the ion balance in the system, so that the hydrogel component has stable gelation properties;

[0028] (2.3) Gelation: Transfer the neutralized solution to a 37°C incubator and let it stand for 25-30 minutes to form a gel. If storage is required, the ungelled sample can be stored at 4°C for no more than one month.

[0029] Preferably, the decellularization solution is prepared by mixing 0.5% SDS and 1% Triton X-100 solution in a volume ratio of 1: 1. The combination of SDS and Triton X-100 can take into account both the removal efficiency of cell components and the retention of matrix components.

[0030] Preferably, in (2.1), the dosage of the adipose decellularized matrix powder, 0.1M HCl solution and pepsin is as follows: 15 mg adipose decellularized matrix powder, 1 ml 0.1M HCl solution, 15 mg pepsin.

[0031] The present invention has the following advantages:

[0032] (1) The present invention uses liquid nitrogen freezing and crushing to evenly crush fat tissue into powder, increase the effective area of ​​the decellularization solution, and significantly improve the thoroughness of decellularization; the composite decellularization solution formed by SDS and Triton X-100 can take into account both the removal efficiency of cell components and the retention of matrix components; in addition, isopropanol is used to further remove fat residues; the cleaning process of the present invention replaces pure water and alcohol treatment multiple times to effectively avoid the problem of fat or decellularization solution residues; the method of the present invention can significantly reduce the immune rejection reaction caused by cell residues and the adverse effects of fat residues on biocompatibility and mechanical properties, thereby improving the applicability of hydrogels in clinical practice.

[0033] (2) During the decellularization process, a mild decellularization solution concentration (0.5% SDS, 1% Triton X-100) and a reasonable treatment time (12 hours) were selected to avoid the damage of key matrix components by high concentration chemical reagents and long-term action;

[0034] The present invention optimizes the ratio of the decellularization solution (0.5% SDS, 1% Triton X-100) and the use time (12 hours), thereby significantly reducing the damage to matrix components such as collagen and glycosaminoglycans, thereby retaining the key biological activity and three-dimensional microstructure of adECM; in addition, during the cleaning process, emphasis is placed on sufficient washing and pure water rinsing to avoid the residue of the decellularization solution in the matrix, further ensuring biological activity and biosafety; compared with traditional high-concentration chemical reagent treatment, the present invention maintains the integrity of the matrix structure while improving the decellularization efficiency, providing a better support effect for subsequent material gelation and tissue repair.

[0035] (3) Enzymatic digestion and precise control of acid-base / ion balance were used. The ratio of powder to digestion solution (15 mg powder: 1 ml 0.1 M HCl) and the amount of enzyme (15 mg pepsin) were optimized to ensure adequate digestion. The pH was further adjusted to 7.3-7.4 and PBS was added to adjust the ion balance, so that the hydrogel component had stable gelation properties.

[0036] Among them, pepsin digestion can efficiently dissolve adECM powder while maintaining the activity of its key components; the present invention also achieves rapid gelation (25-30 minutes) by controlling pH and ion concentration, meeting the operational requirements of filling materials; in addition, the optimized hydrogel material can maintain a stable gel state and can provide sufficient support and stability in tissue repair; compared with the prior art, the hydrogel of the present invention has significant advantages in gelation time and stability, further improving its practicality and reliability in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1This is a rheological characteristic diagram of the adipose decellularized matrix hydrogel material prepared by the present invention;

[0038] Figure 2 This is a comparison diagram of the adipose decellularized matrix hydrogel material of the present invention before and after preparation;

[0039] Figure 3 This is an electron microscope scanning image of the adipose decellularized matrix hydrogel material prepared by the present invention. DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0041] Example 1

[0042] This embodiment relates to a method for preparing adipose decellularized matrix hydrogel material, specifically:

[0043] Step 1, preparation of decellularized matrix powder

[0044] (1.1) Obtain inguinal fat pads from SD rats, wash the adipose tissue with sterile PBS, cut into small pieces, add pure water to cover the tissue, and freeze at -80°C overnight;

[0045] (1.2) The frozen adipose tissue was transferred to a freeze dryer for 48 hours;

[0046] (1.3) Grind into fine powder in liquid nitrogen;

[0047] (1.4) Decellularization: Cells were eluted at 37°C for 12 h using a cell elution solution composed of 0.5% SDS and 1% Triton X-10 in equal volumes. 2.5 ml of decellularization solution was added to each ml of lyophilized fat powder. Residual SDS and Triton X-10 were removed by washing with pure water.

[0048] (1.5) Degreasing treatment: Degreasing treatment was performed with isopropyl alcohol at 37°C for 24 h, followed by washing twice with pure water, then treatment with 75% alcohol for 30 min, and finally rinsing three times with pure water;

[0049] (1.6) The cleaned sample was stored at -80°C overnight and freeze-dried for 48 h to obtain a white translucent powder;

[0050] Step 2, Decellularized Matrix Hydrogel Preparation

[0051] (2.1) Digestion of matrix powder: 300 mg of adECM powder and 30 mg of pepsin were dissolved in 20 ml of 0.1 M hydrochloric acid solution and stirred with a magnetic stirrer for 48 h until no obvious particulate matter remained in the solution;

[0052] (2.2) Adjusting the acid-base / ion balance: Use 1 M NaOH to adjust the pH of the solution to 7.4, then add 10% 10X PBS solution to the solution to balance the ions in the system;

[0053] (2.3) Gelation: The sample was transferred to a 37°C incubator for 25 minutes to obtain a gelled adipose decellularized matrix material.

[0054] Figure 1 Shown is the rheological characteristic diagram of the adipose decellularized matrix hydrogel material prepared by the present invention at 37°C, wherein the storage modulus G' (solid dots) represents the elasticity or energy storage capacity of the material, reflecting its solid-state properties; the consumption modulus G" (hollow dots) reflects the viscosity or energy consumption capacity of the material, representing the liquid properties. When G' exceeds G", it means that the material changes from liquid to solid and forms a gel (i.e., gel point). In the figure, G' rises rapidly after 10 minutes, which means that gelation begins, elasticity gradually increases, and stabilizes after 25 minutes, indicating that a three-dimensional cross-linked network has been formed. G" increases slightly with time, but its value is always significantly lower than G', which shows that the hydrogel is mainly elastic and has high stability, which is beneficial to tissue repair.

[0055] Figure 2 Shown is a comparison of the fat decellularized matrix material prepared by the present invention before and after gelation. As can be seen from the figure, the material is in a liquid state before gelation and has strong fluidity, indicating that a stable cross-linked network structure has not yet been formed inside it. After being treated at 37°C for 25 minutes, the material can gel quickly and effectively, transforming from a liquid state to a solid elastic gel with a more stable appearance and showing significant shape retention ability. This change reflects that the material undergoes chemical or physical cross-linking during the gelation process, transforming it from a viscous liquid to a three-dimensional network structure with elastic properties, reflecting its good gelation ability and being able to provide sufficient support and stability in tissue repair.

[0056] See Figure 3 As shown in the figure, the scanning electron microscope (SEM) image shows the microstructure of the adipose decellularized matrix hydrogel sample, and its surface presents a three-dimensional porous network morphology. The pores are evenly distributed, with a pore size of about 5-10 μm, forming an interconnected pore structure. This network property provides a good channel for cell adhesion, nutrient transport and waste discharge, which is conducive to its application in tissue regeneration and repair. In addition, the fibrous structure of the cross-linked network can be seen in the image, which further shows that the hydrogel has good mechanical strength and elasticity, which is conducive to maintaining its shape stability.

[0057] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. A fat decellularized matrix hydrogel material, characterized in that: It is prepared from freeze-dried fat powder after decellularization, digestion, acid-base balance adjustment and gelation.

2. A method for preparing the adipose decellularized matrix hydrogel material according to claim 1, characterized in that: The following steps are involved: Step 1, preparation of decellularized matrix powder (1.1) Wash and chop the adipose tissue, add pure water to cover the adipose tissue, and freeze at -80℃ overnight; (1.2) Move the frozen adipose tissue to a freeze dryer and freeze dry for 48 hours until the tissue is completely dry; (1.3) Grinding into powder: The freeze-dried tissue was ground into fine powder using a grinding dish in liquid nitrogen and stored at -20°C after processing; (1.4) Decellularization: (a) Preparation of decellularization solution: 0.5% SDS and 1% TritonX-100 solution were mixed; (b) Decellularization: 2.5 ml of decellularization solution was added to each ml of lyophilized fat powder and shaken at 37 °C for 12 h at 250 rpm. (c) Cleaning the decellularization solution: After decellularization, rinse with pure water several times until the sample is clean; (1.5) Degreasing treatment: soak the sample in isopropanol, shake on a shaker at 37°C, then wash twice with pure water, treat with 75% alcohol for 30 min, and rinse three times with pure water; (1.6) The cleaned samples were stored at -80°C overnight and freeze-dried for 48 hours. The powder was white and translucent and was stored at -20°C. Step 2, Decellularized Matrix Hydrogel Preparation (2.1) Matrix powder digestion: Add 0.1 M HCl solution and pepsin to the adipose decellularized matrix powder and stir for 48 h until no visible particles are left; (2.2) Adjusting the acid-base / ion balance: After adjusting the solution pH to 7.3-7.4 with NaOH, add 10% of the total volume of 10X PBS to restore the ion balance in the system; (2.3) Gelation: The neutralized solution was transferred to a 37°C incubator and allowed to stand for 25 to 30 minutes to form a gel.

3. The method for preparing the adipose decellularized matrix hydrogel material according to claim 2, characterized in that: The precondition of the decellularization treatment is: the adipose tissue is freeze-dried and ground into powder and then decellularized.

4. The method for preparing the adipose decellularized matrix hydrogel material according to claim 2, characterized in that: The decellularization solution is prepared by mixing 0.5% SDS and 1% TritonX-100 solution in a volume ratio of 1:

1.

5. The method for preparing the adipose decellularized matrix hydrogel material according to claim 2, characterized in that: In (1.5), the oscillation time is 24 hours.

6. The method for preparing the adipose decellularized matrix hydrogel material according to claim 2, characterized in that: In (2.1), the dosage of the adipose decellularized matrix powder, 0.1M HCl solution and pepsin is as follows: 15 mg adipose decellularized matrix powder, 1 ml 0.1M HCl solution, 15 mg pepsin.

Images