Application of protein-based biological material based on food casing in tissue engineering

Abstract

The invention relates to application of a casing-source protein-based biological material in tissue engineering, and belongs to the technical field of high-value utilization of biological materials. A protein-based biological material is freeze-dried to obtain gel powder, and the gel powder can be directly mixed with a cell suspension for culture, can be directly used as a surgical suture spinning raw material, and can also be doped with other materials to prepare a biological scaffold; the preparation process of the protein-based biological material comprises the following steps: rehydrating, cleaning, freeze-drying and crushing the edible animal casing; under the non-denaturation condition, active protein components are extracted from the smashed animal casings through an enzyme method or an acid method, and the hydrolysis temperature is avoided in the whole extraction process; and adjusting the pH value of the obtained protein component to 7-8 to obtain the protein-based biological material capable of self-assembling under physiological conditions. Compared with the traditional animal-derived collagen, the collagen provided by the invention has the advantages of safe material source, low cost and small difference among batches, and can meet the diversified requirements of different tissue engineering applications on material performance and form.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomaterial preparation and high-value utilization of food by-products, and more particularly relates to an application of a protein-based biomaterial based on food casing in tissue engineering. BACKGROUND

[0002] One of the core challenges of tissue engineering and regenerative medicine is to construct a three-dimensional scaffold that can mimic the microenvironment of the natural extracellular matrix to support cell adhesion, proliferation, differentiation, and the formation of new tissues. Collagen, as the main structural protein of the extracellular matrix, has become an ideal raw material for biological medical materials such as tissue engineering scaffolds, wound dressings, and surgical sutures due to its excellent biocompatibility, low immunogenicity, biodegradability, and inherent cell recognition signals. In particular, native conformational collagen with a complete triple helix structure can self-assemble into a gel under physiological conditions to provide structural support and biological signals for cells, which is a biological function that denatured collagen or small molecule collagen peptides cannot achieve.

[0003] In the field of tissue engineering scaffolds, an ideal protein scaffold should have the following characteristics: (1) the raw material source is sufficient and meets the ethical requirements; (2) the preparation process is mild and can preserve the native structure and self-assembly ability; (3) it can be processed into various forms such as porous scaffolds, hydrogels, and fibers according to the target tissue morphology; (4) it has adjustable mechanical properties and degradation rate; (5) it has excellent biocompatibility and is free of pyrogenic contaminants such as endotoxins. Existing commercial protein scaffolds are mostly derived from bovine tendons, rat tail tendons, etc., which have problems such as limited source, high cost, and the like, which limit their large-scale clinical application.

[0004] It is worth noting that the preparation of existing collagen-based biomaterials mostly relies on extracting collagen from fresh animal tissues (such as cowhide, pigskin, rat tail tendon), which has high cost, long cycle, and involves ethical issues related to animal slaughter. At the same time, the food industry produces a large amount of collagen-rich by-products, such as discarded casings produced during the production of edible animal casings, which are currently mainly treated as low-value waste or converted into small molecule peptides for feed through hydrolysis, but the products completely lose the triple helix structure and self-assembly ability and cannot be used for tissue engineering applications that require structural support functions; a method for preparing functional protein powder from pigskin as raw material, which obtains insoluble powder through physical crushing, is used to replace food additives in meat product processing, but the product is a solid powder, and the collagen protein is still bound in the tissue network and cannot be dissolved to form a processable collagen solution, making it difficult to be used in fields such as scaffold construction and electrospinning that require solution processing.

[0005] In addition, collagen materials used in the biomedical field have strict requirements for endotoxin residues. Endotoxin is a lipopolysaccharide component of the cell wall of gram-negative bacteria, which may cause cytotoxicity, inflammatory response and even implant failure if left in the biomaterial. The existing endotoxin removal methods of tissue-derived collagen often use repeated washing, alkali treatment or chromatographic separation, which often leads to a decrease in collagen yield or structural damage. Developing a processing method that can effectively remove endotoxin while maintaining the triple helix structure and self-assembly ability of collagen protein is a prerequisite for the clinical application of tissue engineering collagen materials.

[0006] In summary, the prior art has the following shortcomings: (1) there is a lack of a preparation method for obtaining active proteins by mild extraction using food industry by-products (waste casings) as raw materials; (2) existing collagen extraction methods have not been effectively integrated with endotoxin removal processes, making it difficult to meet the safety requirements of biomedical materials; (3) the preparation of existing collagen-based tissue engineering products often starts from fresh animal tissues, which is complex and costly, and does not fully utilize low-value by-products; (4) there is no report on the systematic application of waste casing-derived active protein in multiple tissue engineering scenarios such as organoid culture, surgical sutures, wound dressings, and bone cartilage repair scaffolds. Therefore, it is of great scientific significance and industrial value to develop a protein-based biomaterial that uses waste casings as raw materials, has both structure preservation and endotoxin removal capabilities, and can be flexibly processed into various tissue engineering products and their application methods. SUMMARY

[0007] To address the problems of complex traditional collagen extraction processes, high raw material costs, and the failure to fully utilize high-purity edible casing resources in the prior art, the present application provides an application of a protein-based biomaterial in tissue engineering. By crushing the edible animal casing and extracting the protein components under mild conditions, the protein-based biomaterial that can self-assemble into collagen fibers under physiological conditions is obtained after adjusting to physiological pH. The material can be freeze-dried to form a gel powder, which has the characteristics of rapid reconstitution and immediate use, and can be directly mixed with cell suspensions for three-dimensional cell culture, used as a spinning material for surgical sutures, or used to construct composite tissue engineering scaffolds by doping with other biomaterials. The present application converts edible animal casing into a high-value multifunctional biomaterial platform through an efficient, mild process that maximally preserves natural active ingredients, thereby solving the technical problems of high cost, complex process, resource waste, and single application form of existing collagen sources.

[0008] According to a first aspect of the present application, there is provided an application of a casing-derived protein-based biomaterial in tissue engineering, wherein the protein-based biomaterial is prepared by the following steps: (1) rehydrating, washing, freeze-drying and crushing the edible animal casing; (2) The sausage casing pieces of step (1) are extracted under non-denaturing conditions using enzymatic or acid method to obtain an extract containing collagen proteins retaining triple helix structure and other active proteins, and the whole extraction process avoids the hydrolysis temperature of collagen proteins and other active proteins; (3) The protein component obtained in step (2) is adjusted to a pH of 7-8 to obtain a collagen fiber protein-based biomaterial capable of self-assembling to form collagen fibers under physiological conditions.

[0009] Preferably, the application specifically refers to: freeze-drying the protein-based biomaterial to obtain gel powder, mixing the cell suspension directly with the gel powder, and culturing after solidification; or spinning the gel powder as surgical thread; or doping the gel powder with other materials to make tissue scaffolds.

[0010] Preferably, the cell suspension is an organoid suspension, a stem cell suspension, or a cancer cell suspension.

[0011] Preferably, the spinning is electrospinning.

[0012] Preferably, doping the gel powder with other materials specifically refers to: doping the gel powder with hyaluronic acid to make medical adjuvants, or doping the gel powder with bone cement to make cartilage repair materials.

[0013] Preferably, the enzymatic method is extracting under acidic conditions of 0.01-0.1 mol / L HCL solution using pepsin, collagenase or papain, and the final concentration of the pepsin, collagenase or papain is 2-6 mg / mL.

[0014] Preferably, the acid method specifically refers to being performed under the condition of 0.1-0.5 mol / L acetic acid, and the extraction time is 12-48 hours.

[0015] Preferably, between step (1) and step (2), there is also a process of removing endotoxins, specifically: using a solution containing a surfactant to rinse the crushed sausage casing powder, then adding a sucrose solution, and then heating and incubating, the solution changes from clear to turbid, and finally separates into two layers, after the two phases are completely separated and a clear interface is formed, the upper water phase is discarded, and the sausage casing powder with endotoxins removed is obtained.

[0016] Preferably, the surfactant is Triton x-114.

[0017] Preferably, the rinsing is performed at 4°C-6°C.

[0018] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: (1) Create a multifunctional application platform for collagen from sausage casing in tissue engineering: The present invention first systematically applies edible animal sausage casing-derived materials that retain active proteins to various tissue engineering scenarios such as three-dimensional cell culture, surgical suture preparation, and tissue engineering scaffold construction. Compared with traditional animal-derived collagen, the materials in this invention are safe, low-cost, and have small batch-to-batch differences, meeting the diverse needs of material performance and form for different tissue engineering applications.

[0019] (2) Mild process and component preservation: The present invention uses enzyme extraction process (4-25℃) or acid extraction process (4-15℃) combined with endotoxin removal steps to maximize the preservation of the natural triple helix structure and biological activity of collagen. Compared with traditional gelatin extraction process (>100℃), this method avoids the thermal denaturation and degradation of collagen, while retaining the inherent elastin, glycosaminoglycans, and other active ingredients in the sausage casing, making the final product a more complex biomaterial that is closer to the natural extracellular matrix. The biological compatibility and functional diversity of the product are significantly better than those of single-component gelatin or denatured collagen. Collagen with triple helix structure can self-assemble into characteristic fiber networks under physiological conditions, providing a biomimetic microenvironment for cells, which is a biological function that denatured collagen or small molecule peptides cannot achieve.

[0020] (3) Raw material innovation and high-value utilization: The present invention creatively uses standardized edible animal sausage casing (salted / dried sausage casing) as the core raw material, opening up a new and high-quality collagen source. This not only realizes the super-high-value utilization of food processing byproducts, turning waste into treasure, but also provides pure and stable raw materials that meet food-grade safety standards, effectively avoiding market price fluctuations and supply chain risks of traditional gelatin raw materials (pigskin, cow bone), while avoiding ethical controversies and high costs of extracting collagen from fresh animal tissues.

[0021] (4) Convenient product form and flexible application: The present invention creatively processes the gel liquid into a freeze-dried powder form that can be quickly reconstituted. This product has good stability, is easy to store and transport, and achieves the great convenience of "ready-to-use and ready-to-reconstitute". In three-dimensional cell culture applications, users only need to mix the cell suspension directly with the gel powder to quickly obtain a cell-loaded gel system with uniform cell distribution. In surgical suture preparation, the gel powder can be directly used as a static spinning raw material, avoiding the cumbersome process of dissolving and spinning traditional collagen. In tissue engineering scaffold construction, the gel powder can be flexibly doped with other biomaterials to achieve functional synergy of composite materials. This "one powder for multiple uses" design concept significantly improves the convenience and flexibility of the material's use.

[0022] (5) Complete technical path and significant industrialization advantages: This invention provides a complete, efficient, and green technical path from specific waste resources (edible animal casings) to high-end biomaterials (multifunctional tissue engineering products), covering key links such as raw material pretreatment, gentle extraction, endotoxin removal, freeze-drying, and multi-scenario applications. Compared with existing technologies, this invention has significant advantages in reducing raw material costs, simplifying preparation processes, improving product performance, expanding application scope, and ensuring biosafety. It can meet the application needs of multiple fields such as cell culture, tissue repair, and medical devices, and has good industrialization prospects and market competitiveness. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the preparation of the protein-based biomaterial of the present invention.

[0024] Figure 2 This is a histological staining image of the edible sausage casing of the present invention.

[0025] Figure 3 This is a diagram showing the DNA content of the edible casing of this invention.

[0026] Figure 4 This is a diagram showing the glycosaminoglycan content of the edible casing of this invention.

[0027] Figure 5 This is a comparison diagram of organoid growth in Embodiment 1 of the present invention.

[0028] Figure 6 This is a graph showing the relative cell activity in matrix gel and casing gel in Example 1 of the present invention.

[0029] Figure 7 This is a physical image of the surgical suture used in Embodiment 2 of the present invention.

[0030] Figure 8 This is a microstructure diagram of the medical dressing in Embodiment 3 of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Figure 1 This is a schematic diagram illustrating the preparation of the protein-based biomaterial of the present invention.

[0033] This invention uses food-grade edible animal casings, made from... Figure 2Histological staining revealed that the casing did not show purple in hematoxylin-eosin staining, indicating the absence of cellular components; it retained most of the red color in Sirius red staining and most of the blue color in Masson staining, indicating the retention of a large amount of collagen; although the color was lighter in Alcian blue and Alcian blue-periodic acid-Schiff staining, it still showed blue-purple color, indicating the retention of glycosaminoglycans, which requires further quantification.

[0034] This invention uses food-grade edible animal casings, made from... Figure 3 It is known that the DNA content of the casing itself is much lower than that of fresh tissue, and is comparable to that of decellularized fresh tissue.

[0035] This invention uses food-grade edible animal casings. Figure 4 It can be seen that although the content of glycosaminoglycans decreased compared with fresh tissue and decellularized fresh tissue, some were still retained.

[0036] The following are specific examples.

[0037] Example 1: Intestinal coating protein gel for organoid culture 1. Raw materials: Take 1 kg of salted finished pig intestine casings.

[0038] 2. Cleaning: Soak in pure water, scrub and change the water until the water becomes clear.

[0039] 3. Freeze-drying: Freeze the processed sausage casings at -80°C for more than 3 hours, then transfer them to a freeze dryer.

[0040] 4. Grinding: Put the freeze-dried sausage casings into a grinder for grinding.

[0041] 5. Endotoxin removal: Rinse the pulverized sausage casing powder with a solution containing 1% Tritonx-114, stirring gently at 4°C for 60 minutes, changing the solution every 30 minutes. Add a 6% sucrose solution and transfer to a 20°C water bath or incubator for 10-15 minutes. The solution will change from clear to turbid, eventually separating into two layers: the upper layer is a micelle phase (rich in detergent and endotoxin).

[0042] Immediately transfer the test tube to a centrifuge at 20°C and centrifuge at an appropriate speed (e.g., 500-1000 × g) for 10 minutes to accelerate complete separation of the two phases and form a clear interface. Carefully discard the upper aqueous phase using a pipette or tube. Add fresh Triton X-114 to the solid remaining from the previous step and repeat the above process of low-temperature mixing, incubation, centrifugation, and collection. Performing 2-3 rounds can significantly reduce residue.

[0043] 6. Digestion: Pour the powder into 0.02 mol / L HCl, add pepsin (4 mg / mL), and stir at room temperature for 24 h.

[0044] 7. Adjust pH: Adjust the pH of the digested liquid to 7.4 using 0.5M NaOH.

[0045] 8. Place the neutralized gel solution in a freeze dryer at -80°C for more than 3 hours, then transfer it to a freeze dryer to make gel powder.

[0046] 9. Mix 1 mL of digested colorectal cancer organoid suspension with 10 mg of dry gel and seed it into a well plate.

[0047] 10. Cured at 37°C upright for 5 minutes, then inverted for 25 minutes. After adding culture medium, it was placed in an incubator for incubation. The incubation results were compared with those of traditional incubation methods. Figure 5 As shown. Figure 6 This is a graph showing the relative cell activity in matrix gel and casing gel in Example 1 of the present invention. Figure 6 It can be seen that there is no difference in the culture effect between substrate gel and sausage casing gel. Example 2: Preparation of surgical sutures containing gut protein Raw material pretreatment: Take 100g of salted pig casings, thoroughly rehydrate and rub them with deionized water to completely remove surface salt and impurities. Then, soak the washed casings in a 0.1M sodium hydroxide solution at 4℃ for 1 hour, followed by ultrasonic treatment with pure water to effectively remove endotoxins. After treatment, rinse thoroughly with plenty of deionized water until neutral.

[0048] Protein extraction: The pretreated casings were cut into small pieces and placed in a 0.5 M acetic acid solution (solid-liquid ratio 1:30). Extraction was carried out at 4°C with continuous stirring for 72 hours. After extraction, the mixture was centrifuged at 4°C and 8,000 rpm for 20 minutes, and the supernatant was collected.

[0049] Gelation and Shaping: The pH of the supernatant was precisely adjusted to 7.4 using 1 M NaOH solution. Then, genipin at a final concentration of 0.5% (w / v) was added as a crosslinking agent, and after thorough mixing, the mixture was immediately drawn into a dedicated fiber spinning device. By controlling the extrusion rate and the coagulation bath (e.g., a high-concentration ethanol solution), the collagen solution was spun into fine filaments.

[0050] Post-processing and performance: The formed collagen filaments were cured and cross-linked at 37°C for 24 hours, then washed with deionized water and freeze-dried to obtain collagen surgical sutures. Figure 7 This is a physical image of the surgical suture used in Embodiment 2 of the present invention.

[0051] Example 3: Preparation of porous lyophilized gel medical dressing Raw material pretreatment and extraction: Take 50g of dried sheep casing, crush it, and then clean, remove endotoxins and extract it with enzymes according to the method in Example 1 to obtain collagen extract.

[0052] Preparation of pre-gel solution and pouring: Adjust the pH of the extract to 7.0. To enhance its moisturizing and repairing properties, add 5 mg of hyaluronic acid to every 100 mL of collagen solution and stir well. Pour this mixture into a custom mold.

[0053] Crosslinking and freeze-drying: The mold containing the solution was placed in an incubator at 37°C for 1 hour to induce the initial formation of a thermogel. Subsequently, it was immersed in a 0.3% (w / v) genipin solution for secondary crosslinking for 6 hours. After crosslinking, it was rinsed with deionized water and then freeze-dried together with the mold (for easy storage).

[0054] Product and Effects: After demolding, a collagen-hyaluronic acid composite dressing with a porous structure is obtained. Figure 8 This is a microstructure diagram of the medical dressing in Embodiment 3 of the present invention.

[0055] Example 4: Preparation of a cartilage repair scaffold using composite bone cement Preparation of collagen gel powder: Prepare a pH-neutral collagen extract according to the method of Example 1 or 2, freeze-dry it, and grind it to obtain collagen gel powder with uniform particle size distribution.

[0056] Preparation of the composite scaffold: The above-mentioned collagen gel powder and injectable calcium phosphate cement (CPC) powder were physically mixed uniformly under sterile conditions at a mass ratio of 1:4. Before use, the mixed powder was prepared into a paste with the curing solution (phosphate buffer containing 0.05% genipin) according to the manufacturer's recommended ratio.

[0057] Molding and Curing: The paste-like composite was injected into a mold with the anatomical curvature of the cartilage layer and cured for 24 hours at 37°C and 100% humidity. During this process, genipin simultaneously crosslinks the inorganic phases in collagen and CPC, forming an organic-inorganic interpenetrating network.

[0058] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of an intestinal coating-derived protein-based biomaterial in tissue engineering, wherein the protein-based biomaterial is prepared by the following steps: (1) Rehydrate, wash, freeze-dry and pulverize edible animal casings; (2) Extract the casing fragments from step (1) under non-denaturing conditions using enzymatic or acid methods to obtain an extract containing collagen and other active proteins that retain the triple helix structure. The extraction process avoids the hydrolysis temperature of collagen and other active proteins throughout. (3) Adjust the pH of the protein component obtained in step (2) to 7-8 to obtain a collagen fiber protein-based biomaterial that can self-assemble under physiological conditions.

2. The application as described in claim 1, characterized in that, The specific applications are as follows: freezing-drying protein-based biomaterials to obtain gel powder, directly mixing cell suspension with the gel powder, solidifying and then culturing; or using the gel powder as a raw material for surgical suture spinning; or mixing the gel powder with other materials to create tissue scaffolds.

3. The application as described in claim 2, characterized in that, The cell suspension is an organoid suspension, a stem cell suspension, or a cancer cell suspension.

4. The application as described in claim 2, characterized in that, The spinning process is electrospinning.

5. The application as described in claim 2, characterized in that, Specifically, the gel powder is mixed with other materials to produce medical excipients by mixing it with hyaluronic acid, or with bone cement to produce cartilage repair materials.

6. The application as described in claim 1, characterized in that, The enzymatic method involves extraction using pepsin, collagenase, or papain under acidic conditions in a 0.01-0.1 mol / L HCl solution, with the final concentration of the pepsin, collagenase, or papain being 2-6 mg / mL.

7. The application as described in claim 1, characterized in that, The acid method is specifically carried out under acetic acid conditions with a concentration of 0.1-0.5 mol / L, and the extraction time is 12-48 hours.

8. The application as described in claim 1, characterized in that, Between steps (1) and (2), there is also a process for removing endotoxins, specifically: the pulverized casing powder is rinsed with a solution containing surfactant, then sucrose solution is added, and then heated and incubated. The solution changes from clear to turbid and eventually separates into layers. After the two phases are completely separated and a clear interface is formed, the upper aqueous phase is discarded to obtain casing powder with endotoxins removed.

9. The application as described in claim 8, characterized in that, The surfactant is Tritonx-114.

10. The application as described in claim 8, characterized in that, The rinsing is carried out at 4°C-6°C.

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