CN102886063A - Preparation and application of cellulose nanocrystals (CNCs)-reinforced collagen compound substrate - Google Patents

Abstract

The invention discloses a nano-microcrystalline cellulose-reinforced collagen composite matrix, a preparation method and application thereof, and belongs to the field of tissue engineering. The preparation method includes the following steps: adopting the method of degrading cellulose with sulfuric acid to prepare nanocrystalline cellulose and cellulose (CNCs); preparing CNCs solution and collagen solution, and physically mixing the two; then pouring the mixed solution into a mold, After molding, the CNCs-enhanced collagen composite matrix is obtained. The CNCs-enhanced collagen composite matrix obtained by the present invention overcomes the problems of poor mechanical strength and poor hydrophilicity of existing collagen materials, and can be applied to the fields of wound dressings, tissue-induced regeneration membranes, soft tissue patches, tissue-engineered skin and facial masks, etc. . The preparation process of the invention is simple and feasible, has good repeatability, and is suitable for the industrial production of CNCs reinforced collagen composite matrix with different requirements.

Description

Preparation and application of a nano-microcrystalline cellulose-reinforced collagen composite matrix

technical field

The invention belongs to the field of tissue engineering and relates to a collagen matrix, in particular to the preparation and application of a nano-microcrystalline cellulose-reinforced collagen composite matrix.

Background technique

Collagen is the main component of the extracellular matrix and the most abundant structural protein in the human body, accounting for about 25% to 35% of the total protein in the body, mainly found in connective tissue. Because of the high content of collagen, connective tissue has certain structural and mechanical properties, such as tensile strength, tension, elasticity, etc., to achieve the functions of support and protection.

Collagen has good biocompatibility, nutrition, repair, moisturizing, compatibility and affinity, so it is widely used in functional products such as biomedical materials, cosmetics, food and health products. Collagen has the functions of beauty (anti-wrinkle, moisturizing, whitening, weight loss, breast enhancement), preventing osteoporosis, improving joint health, improving blood circulation, strengthening stomach, and improving human immunity.

The skin is the largest organ of the human body, accounting for about 16% of the body weight. It can be divided into three parts: the epidermis, the dermis and the subcutaneous tissue. The dermis is a dense connective tissue with a thickness of about 0.6-3 mm, mainly composed of collagen, elastin, and proteoglycan. The dermis plays an important role in the elasticity and mechanical integrity of the skin. Due to the defect of the dermal layer of the skin caused by various injuries, a large amount of collagen in the tissue is also lost. Therefore, the preparation of various wound dressings and tissue engineered skin using collagen as a raw material has attracted great attention. Already commercialized artificial skin Integra, Apligraft, etc. contain collagen components. Basic studies have shown that providing a suitable moist environment for wounds is beneficial to wound healing. Research has also shown that artificial skin has mechanical properties similar to natural skin and can promote wound healing. Therefore, the new matrix should have similar mechanical properties to human skin while keeping the wound moist. Pure collagen matrix has the advantages of good biocompatibility, easy processing, plasticity, and the ability to promote cell adsorption and proliferation, but it also has poor mechanical properties, is difficult to shape when it contains water, and cannot support tissue reconstruction.

Basic studies have shown that collagen matrix provides a suitable moist environment for wounds, which is beneficial to wound healing. Studies have also shown that collagen matrices have mechanical properties similar to natural skin and can promote wound healing. Therefore, the new matrix should have similar mechanical properties to human skin while keeping the wound moist. Tissue engineered skin prepared from pure collagen matrix has the advantages of good biocompatibility, easy processing, shapeability, and the ability to promote cell adsorption and proliferation.

The aging of collagen in daily life is the main cause of skin wrinkles. The supplement of collagen plays an irreplaceable role in improving the function of aging skin. At present, there are three main methods of collagen supplementation: oral administration, transdermal administration and injection. Among them, oral administration is mainly through food intake and health care products are the main methods. For beauty, facial masks are used transdermally, and injections are mainly used for high-end beauty treatment. Masks are mainly coated and applied, and the coated collagen lasts for a short time, and pure collagen films and composite collagen films are mainly used for application. Due to the high cost of pure collagen membranes, it is particularly important to find composite collagen membranes with excellent performance and low cost.

In recent years, enhancing the mechanical strength of composites with materials with regenerative capabilities has attracted great attention, especially nanocrystalline cellulose (CNCs) because of their renewability, biodegradability and remarkable mechanical strength. performance has become a research hotspot. CNCs are generally prepared from natural cellulose through chemical, biological, physical and other methods. CNCs not only have the basic structure and properties of cellulose but also have some characteristics of nanoparticles, and at the same time have many excellent properties: such as biodegradability, high crystallinity, high strength, high hydrophilicity, high modulus, ultrafine Structure, non-toxic, etc., has broad application prospects in the field of biomedicine.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a method for preparing a nano-microcrystalline cellulose-reinforced collagen composite matrix. Combining collagen and nano-microcrystalline cellulose, two natural organic polymer materials, can not only enhance the strength and biological activity of the material, but also give full play to the bio-nano effect, effectively making up for the limitations of pure collagen in application.

Another object of the present invention is to provide the nano-microcrystalline cellulose-reinforced collagen composite matrix prepared by the above preparation method.

Another object of the present invention is to provide the application of the above-mentioned nano-microcrystalline cellulose reinforced collagen composite matrix.

The object of the present invention is achieved through the following technical solutions: a preparation method of nano-microcrystalline cellulose reinforced collagen composite matrix, comprising the steps of:

(1) Using cellulose as raw material to prepare nanocrystalline cellulose by sulfuric acid hydrolysis.

(2) Prepare a nanocrystalline cellulose solution with a mass concentration of 1% to 10% and a collagen solution with a mass concentration of 1% to 10%.

(3) The above-mentioned nano-microcrystalline cellulose solution and collagen solution are prepared into a nano-microcrystalline cellulose/collagen mixed solution with a mass ratio of nano-microcrystalline cellulose to collagen ≤ 1:10, and the nano-microcrystalline cellulose/collagen mixed solution Pour it into a mold, and get the nano-microcrystalline cellulose-reinforced collagen composite matrix after molding.

The cellulose described in step (1) is plant cellulose or microbial cellulose.

The conditions of the sulfuric acid hydrolysis method described in step (1) are preferably as follows: add 5-15g of cellulose to 90-150mL of concentrated sulfuric acid for reaction, then add deionized water to terminate the reaction, ultrasonically disperse after cooling, and then dialyze to pH It is stable (pH about 7.0), and nanocrystalline cellulose can be obtained after drying. The sulfuric acid is preferably sulfuric acid with a mass concentration of 35% to 64%; the reaction is preferably a mechanical stirring reaction in a constant temperature water bath at 45 to 50°C for 4 to 6 hours; the amount of deionized water is preferably 800 to 1000mL The condition of the ultrasonic dispersion is preferably 25 ~ 35kHz ultrasonic dispersion for 30min; the dialysis is preferably deionized water dialysis; the drying is preferably freeze drying, and the conditions are preferably: at -10 ~ -20 ° C Freeze overnight, and then freeze-dry in a freeze dryer for 12-24 hours.

Both the nanocrystalline cellulose solution and the collagen solution described in step (2) are preferably prepared using acetic acid solution as a solvent; the mass concentration of the acetic acid solution is preferably 0.3%-1%.

The mass concentration of the nanocrystalline cellulose solution described in step (2) is preferably 1%-3%.

The mass concentration of the collagen solution described in step (2) is preferably 1%-3%.

The mixing described in step (3) is preferably performed by mechanical stirring for 1-3 hours.

The material of the mold described in step (3) is preferably glass, stainless steel, solvent-resistant plastic or polytetrafluoroethylene.

The molding method described in step (3) is preferably a solvent evaporation method or a freeze-drying method; the solvent evaporation method is preferably evaporated using an oven at 30-40°C, and the conditions of the freeze-drying method are preferably at Freeze at -10～-20°C overnight, and then freeze-dry in a freeze dryer for 12-24 hours.

A nano-microcrystalline cellulose-reinforced collagen composite matrix is prepared through the above-mentioned preparation method.

The nano-microcrystalline cellulose-reinforced collagen composite matrix can better promote wound healing, and can be applied to fields such as wound dressings, tissue-induced regeneration membranes, soft tissue patches, tissue-engineered skins, and facial masks.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The nano-microcrystalline cellulose-reinforced collagen composite matrix prepared by the present invention is characterized in that it uses nano-microcrystalline cellulose to strengthen the collagen matrix to prepare a composite matrix. By changing the mass percentage and molding method of nano-microcrystalline cellulose, Composite matrices with specific mechanical strength, macrostructure (3D scaffolds, membranes) and microstructure (pore size, porosity, etc.) can be obtained;

(2) The swelling performance test shows that the nano-microcrystalline cellulose reinforced collagen composite matrix has excellent liquid absorption capacity, and the water absorption rate is as high as 500%. The mechanical property test shows that the mechanical strength of the composite matrix is significantly improved compared with the pure collagen scaffold ( 30%); the results of cell experiments showed that the composite matrix can effectively promote the adhesion and proliferation of fibroblasts, and has good cytocompatibility.

(3) The preparation process of the present invention is simple, the source of materials is wide, and the production efficiency is high, which is suitable for the industrial production of nano-microcrystalline cellulose-reinforced collagen composite matrix with different needs.

Description of drawings

Figure 1 is the macroscopic morphology of 5wt%CNCs reinforced collagen composite film.

Figure 2 is the scanning electron micrograph of 5wt%CNCs reinforced collagen composite film, a is the scanning electron micrograph of the surface, and b is the scanning electron micrograph of the cross-sectional morphology.

Fig. 3 is the macroscopic morphology of 10wt% CNCs reinforced collagen composite scaffold.

Figure 4 is the scanning electron micrograph of 10wt% CNCs reinforced collagen composite scaffold, a is the scanning electron micrograph of the surface, and b is the scanning electron micrograph of the cross-sectional morphology.

Fig. 5 is a graph showing the swelling properties of different percentages of CNCs-enhanced collagen composite membranes.

Fig. 6 is the stress-strain diagram of different percentage CNCs reinforced collagen composite membranes.

Figure 7 is the SEM image of fibroblasts (3T3) cultured on 5wt% CNCs reinforced collagen composite membrane for 3 days.

Fig. 8 is a scanning electron micrograph of a composite film prepared without a material mold.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

Add 15g of cotton cellulose to 150mL of 64%w/w concentrated sulfuric acid, in a constant temperature water bath at 45°C, mechanically stir the reaction for 6 hours, add 800mL of deionized water to terminate the reaction, after cooling, ultrasonically (35kHz) disperse for 30min, remove After 3 days of dialysis with ionized water, freeze overnight at -20°C, and then freeze-dry in a freeze dryer for 12 hours to prepare nanocrystalline cellulose (CNCs). Use the 0.3% acetic acid solution as the solvent to prepare the CNCs solution and the collagen solution with a mass concentration of 1%; get 0.5 g of the above CNCs solution and drop it into the 9.5 g of the above collagen solution, and mechanically stir for 2 hours to obtain the CNCs solution with a content of 5% w/w CNCs/collagen mixed solution, then pour the mixed solution into a polytetrafluoroethylene mold, put it in an oven at 35°C, and make a 5wt% CNCs-reinforced collagen composite film after the solvent is evaporated and formed. The macroscopic morphology of the CNCs reinforced collagen composite film is shown in Figure 1, and the scanning electron microscope images of its surface and cross-sectional morphology are shown in Figure 2.

Example 2

Rinse the commercially available bacterial cellulose with clean water several times to remove the culture medium and impurities on the surface of the membrane, then soak the membrane in 0.5mol/L NaOH solution, cook at 90°C for 20min or more, cool, place in deionized water and soak repeatedly until Neutral, bacterial cellulose solid was obtained after freeze-drying. Take 5g of dried bacterial cellulose, add it to 100mL of sulfuric acid with a mass percentage of 35%, react in a constant temperature water bath at 50°C for 4 hours, add 800mL of deionized water to terminate the reaction, and after cooling, disperse with ultrasonic (35kHz) for 30min, and dialyze with deionized water for 3 days After drying, the nanocrystalline cellulose (cellulose nanocrystals, CNCs) is obtained. Use acetic acid solution with a mass concentration of 1% as a solvent to prepare CNCs solution and collagen solution with a mass concentration of 3%; take 1g of the above CNCs solution and drop it into 9g of the above collagen solution, and mechanically stir for 2 hours to obtain a CNCs solution with a content of 10%. w/w CNCs/collagen mixed solution, and then the mixed solution was added to a stainless steel mold, frozen overnight at -10°C, and then placed in a freeze dryer for 36 hours to obtain a 10wt% CNCs-reinforced collagen composite scaffold. The macroscopic topography of the CNCs-reinforced collagen composite scaffold is shown in Figure 3, and the scanning electron micrographs of its surface and cross-sectional topography are shown in Figure 4.

Example 3

The method of degrading cotton cellulose with sulfuric acid in Example 1 was used to prepare nanocrystalline cellulose (cellulosenanocrystals, CNCs). Use acetic acid solution with a mass concentration of 0.5% as a solvent to prepare CNCs solution and collagen solution with a mass concentration of 2%; respectively take different volumes of the above CNCs solution and drop them into the above collagen solution, and mechanically stir for 2 hours to obtain the CNCs solution. 1wt%, 3wt%, 5wt%, 7wt% and 10wt% CNCs/collagen mixed solution, then pour the mixed solution into a polytetrafluoroethylene mold, put it in an oven at 40°C, and evaporate the solvent to make 1wt %, 3wt%, 5wt%, 7wt% and 10wt% CNCs reinforced collagen composite membranes. The swelling rate of the composite membrane was measured by weighing method, and the results are shown in Figure 5. The mechanical strength testing machine (Instron5543) was used to measure the stress-strain curve of the composite membrane with 5 parallel samples in each group, and the results are shown in Figure 6.

Sterilize the above series of CNCs-enhanced collagen composite membranes with 75% ethanol, put them on an ultra-clean table to dry, then put 1 mL of culture medium into a 24-well plate overnight, absorb the culture medium the next day, and add 5 ×10 ⁴ fibroblasts (3T3), adding culture medium for 1, 3, 5, 7 days to investigate the adhesion and proliferation behavior of 3T3 cells on different membranes, the results show that the collagen composite membrane enhanced by CNCs can effectively promote fibrogenesis Adhesion and proliferation of cells, with good cytocompatibility. Figure 7 is a SEM image of 3T3 fibroblasts cultured on the surface of 5wt% CNCs enhanced collagen composite membrane for 3 days.

Example 4

The method of degrading cotton cellulose with sulfuric acid in Example 1 was used to prepare nanocrystalline cellulose (cellulosenanocrystals, CNCs). Use acetic acid solution with a mass concentration of 0.3% as a solvent to prepare CNCs solution and collagen solution with a mass concentration of 1%; take 0.3mL of the above CNCs solution and drop it into 9.7mL of the above collagen solution, and mechanically stir for 2 hours to obtain the content of the CNCs solution. It is a 3% CNCs/collagen mixed solution, and then the mixed solution is poured into molds of different materials, put into an oven at 30°C, and the 3wt% CNCs-reinforced collagen composite film is obtained after the solvent is evaporated and formed. Observation by electron microscope (Figure 8 shows the scanning electron micrographs of composite films prepared by molds of different materials, a polytetrafluoroethylene mold, b glass mold, c stainless steel mold), it can be seen that compared with glass molds and polytetrafluoroethylene molds, stainless steel The collagen composite membrane prepared by the mold has larger pores; the surface of the composite membrane prepared by the polytetrafluoroethylene mold is relatively flat and smooth.

Example 5

Rinse the commercially available bacterial cellulose with clean water several times to remove the culture medium and impurities on the surface of the membrane, then soak the membrane in 0.5mol/L NaOH solution, cook at 90°C for 20min or more, cool, place in deionized water and soak repeatedly until Neutral, bacterial cellulose solid was obtained after freeze-drying. Take 5g of dried bacterial cellulose, add it to 100mL of sulfuric acid with a mass percentage of 35%, react in a constant temperature water bath at 50°C for 4 hours, add 800mL of deionized water to terminate the reaction, and after cooling, disperse with ultrasonic (35kHz) for 30min, and dialyze with deionized water for 3 days After drying, the nanocrystalline cellulose (cellulose nanocrystals, CNCs) is obtained. Use the 1% acetic acid solution as the solvent to prepare the CNCs solution and the collagen solution with a mass concentration of 3%; get 0.5 g of the above CNCs solution and drop it into the 9.5 g of the above collagen solution, and mechanically stir for 2 hours to obtain the CNCs solution with a content of 5wt% CNCs/collagen mixed solution, and then the mixed solution was added to a stainless steel mold, frozen overnight at -20°C, and then placed in a lyophilizer for 36 hours to obtain a 5wt% CNCs-reinforced collagen composite scaffold. The above-mentioned composite brackets were sterilized with 75% ethanol, placed on an ultra-clean table to dry, and set aside. The composite scaffold was implanted into the back wound model of rats, and the in vivo experimental results showed that the composite scaffold could promote wound healing.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. the preparation method of a nano micro crystal cellulose enhancing collagen composite substrate is characterized in that comprising the steps:

(1) prepares nano micro crystal cellulose take cellulose as raw material by sulphuric acid hydrolysis;

(2) the preparation mass concentration is that 1%～10% nano micro crystal cellulose solution and mass concentration are 1%～10% collagen solution;

(3) above-mentioned nano micro crystal cellulose solution and collagen solution are mixed with the nano micro crystal cellulose of nano micro crystal cellulose and collagen mass ratio≤1:10/collagen mixed solution, nano micro crystal cellulose/collagen mixed solution is poured in the mould, namely obtained nano micro crystal cellulose after the molding and strengthen collagen composite substrate.

2. nano micro crystal cellulose according to claim 1 strengthens the preparation method of collagen composite substrate, it is characterized in that:

Cellulose described in the step (1) is plant cellulose or micro organism cellulose;

The condition of the sulphuric acid hydrolysis described in the step (1) is: 10～15g cellulose is joined in 100～150mL concentrated sulphuric acid react, then add the deionized water cessation reaction, ultra-sonic dispersion after the cooling is dialysed stable to pH again, namely obtains nano micro crystal cellulose after the drying.

3. nano micro crystal cellulose according to claim 2 strengthens the preparation method of collagen composite substrate, it is characterized in that:

Described sulphuric acid is that mass concentration is 35%～64% sulphuric acid; Described reaction is mechanical agitation reaction 4～6h under 45～50 ℃ of waters bath with thermostatic control; The amount of described deionized water is 800～1000mL; The condition of described ultra-sonic dispersion is 25～35kHz ultra-sonic dispersion 30min; Described dialysis is for using the deionized water dialysis; Described drying is lyophilization, and its condition is :-10～-20 ℃ of lower freeze overnight, then put into the freeze dryer lyophilizing 12～24 hours.

4. nano micro crystal cellulose according to claim 1 strengthens the preparation method of collagen composite substrate, it is characterized in that:

Nano micro crystal cellulose solution described in the step (2) and collagen solution all use acetic acid solution to prepare as solvent;

The mass concentration of the nano micro crystal cellulose solution described in the step (2) is 1%～3%;

The mass concentration of the collagen solution described in the step (2) is 1%～3%.

5. nano micro crystal cellulose according to claim 4 strengthens the preparation method of collagen composite substrate, it is characterized in that:

The mass concentration of described acetic acid solution is 0.3%～1%.

6. nano micro crystal cellulose according to claim 1 strengthens the preparation method of collagen composite substrate, it is characterized in that:

Mixing described in the step (3) is for passing through mechanical agitation 1～3h mixing;

The material of the mould described in the step (3) is glass, rustless steel, the plastics of anti-solvent the or politef;

The method of the molding described in the step (3) is solvent evaporated method or freeze-drying.

7. nano micro crystal cellulose according to claim 6 strengthens the preparation method of collagen composite substrate, it is characterized in that:

Described solvent evaporated method is for using 30～40 ℃ baking oven to evaporate;

The condition of described freeze-drying is :-10～-20 ℃ of lower freeze overnight, then put into the freeze dryer lyophilizing 12～24 hours.

8. a nano micro crystal cellulose strengthens collagen composite substrate, it is characterized in that: prepared by each described preparation method of claim 1～7.

Nano micro crystal cellulose claimed in claim 8 strengthen collagen composite substrate at wound dressing, organize the application in regeneration induction film, soft-tissue patch, organization engineering skin and/or the facial film field.

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