阅读说明：本技术 基于静电纺丝的丝素蛋白/胰腺脱细胞基质混合支架 (Silk fibroin/pancreatic acellular matrix mixed scaffold based on electrostatic spinning ) 是由 朱逸 郭益冰 王东芝 陆玉华 姚曦浩 邱洪权 黄* 李晓红 缪海燕 吴迪 王志伟 于 2020-05-19 设计创作，主要内容包括：基于静电纺丝的丝素蛋白/胰腺脱细胞基质混合支架,属于纳米生物医用材料及胰腺组织工程领域；丝素蛋白溶解于六氟异丙醇(HFIP),配制浓度为20％(w/v)的丝素蛋白溶液(溶液A)；胰腺脱细胞基质溶解于HFIP,配制浓度为0.4％(w/v)的胰腺脱细胞基质溶液(溶液B)。将溶液A、B混合制备静电纺丝溶液,通过调整静电纺丝参数：混合液中A和B的比例、电压、接收距离、注射器推进速度等获得纤维尺度近似与在体细胞外基质(ECM)纳米结构的薄膜支架,能够为胶原酶分离提取过程导致的细胞外基质破坏的胰岛提供理想的ECM微环境,促进其存活及功能的发挥。(A silk fibroin/pancreatic acellular matrix mixed scaffold based on electrostatic spinning belongs to the field of nano biomedical materials and pancreatic tissue engineering; dissolving silk fibroin in Hexafluoroisopropanol (HFIP) to prepare a silk fibroin solution (solution A) with the concentration of 20% (w/v); the pancreatic acellular matrix was dissolved in HFIP to prepare a pancreatic acellular matrix solution (solution B) having a concentration of 0.4% (w/v). Mixing the solution A, B to prepare an electrostatic spinning solution, and adjusting electrostatic spinning parameters: the membrane scaffold with the fiber size similar to that of an extracellular matrix (ECM) nanostructure is obtained by the proportion, voltage, receiving distance, injector propulsion speed and the like of A and B in the mixed solution, an ideal ECM microenvironment can be provided for pancreatic islets damaged by the extracellular matrix caused by the collagenase separation and extraction process, and survival and function of the pancreatic islets are promoted.)

1. The silk fibroin/pancreatic acellular matrix mixed scaffold based on electrostatic spinning is characterized in that the mixed scaffold is formed by binary compounding of a pancreatic acellular matrix and silk fibroin.

2. The hybrid stent of claim 1, wherein the hybrid stent is prepared by a method comprising:

step 1, passing the silk through Na₂CO₃Removing sericin from the solution, dissolving in EtOH-CaCl₂-H₂Loading into a dialysis bag with molecular weight cut-off of 8-14kD, dialyzing with deionized water with conductivity less than 0.8 μ s/cm for 3 days, freeze drying, and dissolving in hexafluoroisopropanol to obtain solution A;

step 2, oscillating fresh pig pancreas through 1% TritonX-100/0.1% ammonia water to remove cells, washing the fresh pig pancreas through PBS solution to remove residual solvent, freeze-drying the pig pancreas, grinding the pig pancreas into powder through liquid nitrogen, weighing a certain mass of the pig pancreas, and dissolving the pig pancreas in hexafluoroisopropanol to prepare solution B;

and 3, mixing the solution A and the solution B in proportion to prepare the electrostatic spinning solution.

3. The method of preparing a hybrid stent according to claim 2, wherein the mixing ratio of the solution a to the solution B is 60: 1-20: 1 w/w.

4. The method for preparing a hybrid scaffold according to claim 2 or 3, wherein the electrospinning liquid preparation process parameter characteristics are prepared by: mixing the solution A and the solution B, adding the mixed solution A and the mixed solution B into a microinjector controlled by an injection pump, setting the voltage of a high-voltage generator to be 15-25 Kv, the collection distance to be 10-20 cm, the liquid feeding speed to be 1-7 ml/h, receiving and obtaining nano-scale fibers by using a small glass slide adhered to a tin foil paper, then placing the small glass slide into absolute ethyl alcohol for treatment to enable the small glass slide to generate beta folding, and finally placing the small glass slide into air for natural drying for later use.

5. The method of preparing a hybrid stent according to claim 2, wherein the optimal mixing ratio of the solution a to the solution B is 30: 1.

6. the method of claim 4, wherein the high voltage generator has an optimum voltage of 25Kv, the collection distance is preferably 15cm, and the liquid delivery rate is preferably 4 ml/h.

7. The electrospun-based silk fibroin/pancreatic acellular matrix hybrid scaffold according to claim 1, characterized in that the hybrid scaffold can serve as an ideal source of biological nanomaterials in pancreatic islet function protection, pancreatic organoid construction.

Technical Field

The invention belongs to the field of nano biomedical materials and pancreatic tissue engineering, and particularly relates to a method for preparing a silk fibroin/pancreatic acellular matrix hybrid scaffold based on an electrostatic spinning technology, which can be used for protecting the long-term stable exertion of pancreatic islet functions in the process of separating and extracting pancreatic islets by collagenase and other enzymes through simulating an in vivo extracellular matrix microenvironment.

Background

Diabetes mellitus is a genetically predisposed group of metabolic diseases characterized by elevated blood glucose due to insufficient or defective insulin secretion. According to the international diabetes union (IDF) version 8 diabetes atlas 2017, nearly 5 million people worldwide suffer from diabetes, which causes a large economic burden to low-income and medium-income countries and people. China has a large population, and with the improvement of living standard and the diversification of life style, the incidence rate and complication rate of diabetes mellitus increase year by year. Effective and economic treatment means are found, and the comprehensive rehabilitation significance is profound for implementing the Chinese strategy and eliminating the poverty caused by diseases.

The currently commonly used drugs for treating diabetes mainly comprise sulfonylureas, biguanides, alpha-glucosidase inhibitors, insulin sensitivity enhancers and insulin.

However, long-term administration of drugs causes poor compliance, inevitably causes various complications, and often causes hypoglycemia due to inaccurate blood glucose regulation, so that drug therapy is often the palliative method. The most desirable approach is islet transplantation, however, donor source is in short supply and enzymes used in isolation and extraction processes cause extracellular matrix loss and microvascular destruction, leading to impaired islet function or death, further exacerbating donor shortage. Therefore, ensuring stable survival and continuous function of islets after islet harvest is a critical limitation for improving islet transplantation efficiency and success. In recent years, the material for protecting the pancreatic islet function based on hydrogel is rapidly developed, but the hydrogel material cannot well simulate the nano structure of the natural in-vivo extracellular matrix structurally, and the electrostatic spinning technology can produce nano structure fibers similar to the microenvironment of the in-vivo extracellular matrix, is convenient to operate and can be massively produced. Natural polymers, synthetic polymers and acellular matrices can be used alone or in combination for electrospinning preparation. The natural polymer and the synthetic polymer have single composition, and cannot simulate the physiological composition of the extracellular matrix in a high simulation manner even through multi-component compounding. The extracellular matrix of specific tissue and organ has optimal composition, structure and physical and chemical performance corresponding to the functions of cell growth, proliferation, differentiation, etc. Therefore, the invention combines silk fibroin to provide ideal mechanical support, combines the composition of the islet in vivo microenvironment, namely pancreas specific extracellular matrix, forms stable performance, realizes high-simulation islet microenvironment construction, and plays a role in promoting the long-term and stable exertion of islet functions and improving clinical application efficiency. The natural biomaterial is combined with the specific acellular matrix, and a novel electrostatic spinning composite bracket product with the simulated islet extracellular matrix component and structure is successfully developed through the advantages superposition and the defect complementation, so that the novel electrostatic spinning composite bracket product has a wide market prospect.

Disclosure of Invention

The invention mainly solves the technical problems in the prior art and provides the silk fibroin/pancreatic acellular matrix mixed scaffold based on electrostatic spinning.

The technical problem of the invention is mainly solved by the following technical scheme: the silk fibroin/pancreatic acellular matrix mixed scaffold is based on an electrostatic spinning technology, and is formed by binary compounding of a pancreatic acellular matrix and silk fibroin.

Preferably, the preparation method of the hybrid scaffold comprises the following steps:

step 1, passing the silk through Na₂CO₃Removing sericin from the solution, dissolving in EtOH-CaCl₂-H₂Loading into a dialysis bag with molecular weight cut-off of 8-14kD, dialyzing with deionized water with conductivity less than 0.8 μ s/cm for 3 days, freeze drying, and dissolving in hexafluoroisopropanol to obtain solution A;

step 2, oscillating fresh pig pancreas through 1% TritonX-100/0.1% ammonia water to remove cells, washing the fresh pig pancreas through PBS solution to remove residual solvent, freeze-drying the pig pancreas, grinding the pig pancreas into powder through liquid nitrogen, weighing a certain mass of the pig pancreas, and dissolving the pig pancreas in hexafluoroisopropanol to prepare solution B;

and 3, mixing the solution A and the solution B according to a certain proportion to prepare the electrostatic spinning solution.

Preferably, the mixing ratio of the solution A to the solution B is 60: 1-20: 1(w/w), and the optimal mixing ratio is 30: 1.

preferably, the parameter characteristics of the preparation process of the electrospinning solution are prepared by the following method: mixing the solution A and the solution B, adding the mixed solution A and the mixed solution B into a microinjector controlled by an injection pump, setting the voltage of a high-voltage generator to be 15-25 Kv, preferably 25Kv, the collection distance to be 10-20 cm, preferably 15cm, the liquid feeding speed to be 1-7 ml/h, preferably 4ml/h, receiving the nano-scale fibers by using a small glass slide adhered to a tin foil paper, then placing the small glass slide in absolute ethyl alcohol for treatment to enable the small glass slide to generate beta folding, and finally placing the small glass slide in the air for natural drying for later use.

Preferably, the mixed scaffold can be used as an ideal source of biological nano materials in pancreatic islet function protection and pancreas organoid construction.

The invention has the following beneficial effects:

(1) the silk fibroin/pancreatic acellular matrix mixed scaffold prepared by the invention has high simulated pancreatic islet extracellular matrix components and structure, is simple in preparation method, can be spun immediately after use, is short in time consumption and low in cost, can exert the greatest value for the application of pancreatic islet donors with limited sources, and has a good clinical transformation application prospect.

(2) The prepared mixed bracket can obviously improve the survival of islet cells and promote the function of the islet cells.

Drawings

FIG. 1 is a diagram of one nanofiber structure of a silk scaffold and a hybrid scaffold;

FIG. 2 is a comparison of staining of live and dead cells according to the present invention;

FIG. 3 is a comparative graph of an experiment of glucose stimulated insulin release experiments according to the present invention.

Detailed Description

The technical scheme of the invention is further specifically described by the following embodiments and the accompanying drawings.