CN116942917A - In-situ biomineralization-enhanced nanocellulose scaffold and preparation method thereof - Google Patents
In-situ biomineralization-enhanced nanocellulose scaffold and preparation method thereof Download PDFInfo
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Abstract
The application belongs to the field of tissue engineering material creation, and in particular relates to an in-situ biomineralization-enhancing nanocellulose scaffold and a preparation method thereof, wherein the preparation method comprises the following steps: vacuum suction filtration is conducted to induce nano-cellulose deposition to construct a nano-cellulose orientation arrangement film; solvent swelling supported urease; in situ biomineralization constructs anisotropic scaffolds. The application solves the defects of poor mechanical property and limited controllable range of the conventional nanocellulose scaffold material, potential cytotoxicity of the conventional chemical crosslinking enhancer and the like. The method for enhancing the mechanical property of the nano cellulose anisotropic scaffold material by in-situ biomineralization can simultaneously regulate and control the mechanical property of the scaffold material and the scaffold network structure, maintain good biocompatibility, and can meet the double requirements of the tissue engineering material on the structure and the function in biocompatibility. The application has important leading significance for development of tissue engineering materials and biomedical application of nanocellulose.
Description
Technical Field
The application belongs to the field of tissue engineering material creation, and in particular relates to an in-situ biomineralization-enhancing nanocellulose scaffold and a preparation method thereof.
Background
Nanocellulose is a natural cellulose-based material of one-dimensional size on the nanoscale, isolated and extracted from plants (e.g., lignocellulose, seaweed, etc.), animal tissues (e.g., tunicate ascidians), or produced by metabolic secretion of bacteria of a specific species (e.g., acetobacter xylinum). Nanocellulose is mainly divided into three categories, cellulose Nanocrystals (CNCs), cellulose Nanofibers (CNFs), bacterial Cellulose (BC), according to raw material sources and preparation methods. The nanocellulose has chemical components consistent with those of natural cellulose, and is safe and nontoxic. The cellulose raw material is subjected to micro-nano treatment to prepare nano cellulose, and polar groups such as larger specific surface area, surface hydroxyl, carboxyl and the like are further exposed, so that the nano cellulose is endowed with better biocompatibility. The nanocellulose hydrogel, in particular to the nanocellulose (TOCNF and TOBC) prepared by combining TEMPO catalytic oxidation with high-pressure homogenization, is easy to form a three-dimensional network environment with high hydration and swelling in the preparation process, and is highly similar to the collagen network structure of the extracellular matrix of human tissue, thereby providing a good 3D microenvironment for cell adhesion, growth and propagation. Therefore, nanocellulose, in particular TOCNF and TOBC, has shown great potential in biomedical fields of tissue engineering, wound repair, organoid culture, etc.
However, research into constructing tissue engineering materials from nanocellulose still faces many problems. Research shows that the mechanical property, network structure and topology of tissue engineering scaffold material, especially the micro-environment of micro-to nano-level anisotropically arranged scaffold or matrix structure, can directly affect cell morphology and cell shape. The mechanical properties and network structure of the scaffold material are powerful tools for maintaining cell phenotype and inducing cell directed differentiation. However, currently, most hydrogels, including conventional TOCNF and toc, have poor mechanical properties, and their three-dimensional network skeleton is also an isotropic disordered structure in nature, lacking anisotropic structures and excellent biomechanical properties on a microscopic scale of natural tissues like muscle, cartilage, heart valves, etc. Although researchers propose that the mechanical properties of the nanocellulose scaffold material can be regulated and controlled by adding an organic/inorganic reinforcing agent or adopting chemical crosslinking and other means, the controllable range is limited, and the reinforcing agent or the crosslinking agent has the defects of potential cytotoxicity and the like. And the literature reports that the mechanical property and the stent network structure of the stent material can not be regulated and controlled at the same time, and good biocompatibility can be maintained. Therefore, how to construct a network microenvironment with a nanocellulose anisotropic structure and controllable mechanical properties according to the requirements of different organ tissues on the structure and the function of the scaffold material so as to meet the double requirements of the tissue engineering material on the structure and the function in biocompatibility is a challenge in the application of constructing the tissue engineering material by nanocellulose.
Disclosure of Invention
In order to solve the defects existing in biomedical applications of the traditional nanocellulose-based biological scaffold and realize the aim of developing a network microenvironment with a nanocellulose anisotropic structure and controllable mechanical properties, the application adopts the following technical scheme:
the preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold comprises the following steps:
s1, diluting nano cellulose with deionized water, carrying out suction filtration on the nano cellulose diluted solution with a hydrophobic PVDF film under a vacuum condition to form a film, wherein the vacuum acting force, the nano fiber surface charge repulsive force and the gravity are balanced in the suction filtration, so that the nano fiber is induced to be oriented, arranged and deposited layer by layer to form a filter cake;
s2, performing suction filtration until the volume of a filter cake is reduced to 1/50 of the volume of the initial filtrate, stopping the suction filtration, rapidly transferring the filter cake and covering the filter cake with a new hydrophobic PVDF membrane, placing the filter cake between water-absorbing papers, compressing and dehydrating until the filter cake is dried, and replacing the water-absorbing papers every 3 hours until the filter membrane is separated from the PVDF membrane, thereby obtaining the nanocellulose membrane; filtering until the volume of the filter cake is reduced to be higher than 1/50 of the volume of the initial filtrate, and the water content in the filter cake is too high, so that the anisotropic structure is easily damaged in the press drying process; the filter cake volume is reduced to be lower than 1/50 of the initial filtrate volume after the nanocellulose is pressed dry, the nanocellulose is easy to adhere to the PVDF membrane and is not easy to separate;
s3, immersing the nano cellulose film prepared in the step S2 into a swelling liquid containing urease to swell the film and absorb the urease into a nano cellulose hydrogel network, and simultaneously realizing the anisotropic hydrogel lamellar structure construction induced by swelling of the film under the interaction of electrostatic repulsive force on the surface of the nano cellulose and hydrophilic groups on the surface and water molecules;
s4, washing the nano cellulose bracket loaded with the urease in the step S3 by deionized water to remove free urease, and immersing the hydrogel into mineralized liquid to complete the in-situ biomineralization process.
As a preferable technical scheme of the preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold, in the step S1, nanocellulose is TOBC or TOCNF; wherein the TOBC has a diameter of 10-20nm, a length of 500-1000nm and a charge density of 1.0mmol/g; the TOCNF has a diameter of 5-10nm, a length of 200-500nm and a charge density of 1.1mmol/g.
As a preferred technical scheme of the preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold, in step S1, 50mg of TOBC or TOCNFs with dry weight is diluted to 0.1wt% by deionized water, and then a hydrophobic PVDF membrane with a pore diameter of 0.22 μm and a diameter of 47mm is subjected to suction filtration under a suction filtration condition with a vacuum of 10mbar to form a membrane.
As a preferred technical scheme of the preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold, in step S2, the pressure of compression dehydration is 88mbar.
As a preferable technical scheme of the preparation method of the in-situ biomineralization-enhancing nano-cellulose scaffold, in the step S3, the nano-cellulose film is immersed into a swelling solution containing urease for 12 hours at the temperature of 4 ℃, wherein the concentration of the urease in the swelling solution is 10U/mL.
As a preferable technical scheme of the preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold, the swelling liquid is D-PBS, and the concentration of the swelling liquid is 0 to 100vol%.
As a preferable technical scheme of the preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold, in the step S4, the mineralization liquid is urea-CaCl 2 Mixed solution, urea-CaCl 2 The volume ratio of the mixed solution is 1:1 and a molar ratio of urea solution to CaCl of 0.25:1 to 1:0.25 2 The solution is prepared.
As a preferable technical scheme of the preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold, the concentration of the mineralized solution is 0.1mol/L to 1.1mol/L.
Another application is that: the nano cellulose stent prepared by the preparation method has anisotropy in Young modulus in the transverse direction (I) and the axial direction (A), and the mechanical property of the stent can be controlled within the range of 10-1000 kPa.
The beneficial effects of the application are as follows: the application solves the defects of poor mechanical property and limited controllable range of the conventional nanocellulose scaffold material, potential cytotoxicity of the conventional chemical crosslinking enhancer and the like. The method for enhancing the mechanical property of the nano cellulose anisotropic scaffold material by in-situ biomineralization can simultaneously regulate and control the mechanical property of the scaffold material and the scaffold network structure, maintain good biocompatibility, and can meet the double requirements of the tissue engineering material on the structure and the function in biocompatibility. The application has important leading significance for development of tissue engineering materials and biomedical application of nanocellulose.
Drawings
FIG. 1 is the anisotropy index of the swelling medium-controlled stent in step 3 of example 1;
FIG. 2 shows the mechanical properties and mechanical anisotropies of the scaffold controlled by different mineralization process conditions in the step 4 of the embodiment 2;
FIG. 3 is a schematic illustration of example 3 step 4 different urea-CaCl 2 TGA analysis of in-situ biomineralization deposition calcium carbonate under mixed solution molar ratio conditions;
FIG. 4 is a graph showing the cell viability assay on scaffolds of example 4, step 4, different mineralization conditions;
FIG. 5 is a cross-sectional scanning electron microscope image of the in situ biomineralization C-TOBC and C-TOCNF scaffolds of example 4, step 4;
FIG. 6 is a graph showing the expression level of ALP in the in situ biomineralization scaffold cultured stem cells of example 5;
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The raw materials in the application are as follows: nanocellulose includes TOBC (diameter 10-20nm, length 500-1000nm, charge density 1.0 mmol/g) and TOCNF (diameter 5-10nm, length 200-500nm, charge density 1.1 mmol/g) derived from Hainan optical space and Ningbo glycomer, respectively.
Example 1:
s1, diluting TOBC or TOCNFs with dry weight of 50mg to 0.1wt% by deionized water, and then carrying out suction filtration to form a membrane by using a hydrophobic PVDF membrane with a pore diameter of 0.22 mu m (Millipore) and a diameter of 47mm under the suction filtration condition with a vacuum degree of 10mbar, thereby inducing the orientation arrangement of nanofibers to be deposited into a filter cake layer by layer;
s2, performing suction filtration until the volume of a filter cake is reduced to 1/50 of the volume of the initial filtrate, stopping the suction filtration, rapidly transferring a filter membrane and covering the filter membrane with a new hydrophobic PVDF membrane, placing the filter membrane between water-absorbing papers, compressing and dehydrating the water-absorbing papers under the pressure of 88mbar until the water-absorbing papers are dried, and replacing the water-absorbing papers every 3 hours until the filter membrane is separated from the PVDF membrane, thereby obtaining TOBC and TOCNFs films;
s3, immersing the TOBC and TOCNFs film prepared in the step S2 into a D-PBS solution containing urease for 12 hours at the temperature of 4 ℃, swelling the film, and sucking the urease into a nanocellulose hydrogel network to realize the anisotropic hydrogel lamellar structure construction induced by the swelling of the film, wherein the concentration of the urease in the D-PBS solution containing the urease is 10U/mL.
The swelling medium-regulated bracket anisotropy index is monitored by regulating the concentration of swelling liquid D-PBS (0 vol%, 1vol%, 2vol%, 3vol%, 4vol%, 5vol%, 10vol%, 15vol%, 20vol%, 25vol%, 50vol% and 100 vol%), and the specific steps are as follows: the concentration of swelling liquid D-PBS is regulated, the thickness direction (axial direction A) and the diameter direction (transverse direction I) of the hydrogel are monitored, and the ratio of the axial dimension to the transverse dimension is calculated to obtain the anisotropy index of the swelling medium regulating and controlling bracket, wherein the specific numerical values of the anisotropy index are shown in figure 1.
The nanocellulose hydrogel anisotropy index prepared in example 1 is shown in fig. 1, the TOCNFs hydrogel anisotropy index and the TOCNFs hydrogel anisotropy index decrease with increasing D-PBS concentration, respectively, and the TOCNFs hydrogel anisotropy index is higher than the TOCNFs hydrogel. However, when the concentration of D-PBS is 0% by volume (pure deionized water), TOCNFs hydrogel after 24 hours of swelling is completely destroyed in structure due to extremely low mechanical properties, and the structure of a gel lamellar structure is extremely easy to fall off and deform in the process of transferring or touching the gel, so that the size of the TOCNFs hydrogel cannot be accurately measured. In conclusion, researches show that the swelling degree of the hydrogel can be adjusted by selecting the ionic strength of the swelling medium, so that the nanocellulose scaffold material with proper orientation structure and mechanical property can be obtained for subsequent cell culture work.
Example 2
S1, diluting 50mg of TOCNFs (total organic carbon nanotubes) by dry weight to 0.1wt% by deionized water, and then carrying out suction filtration to form a membrane by using a hydrophobic PVDF membrane with the pore diameter of 0.22 mu m (Millipore) and the diameter of 47mm under the suction filtration condition of 10mbar of vacuum degree, thereby inducing the alignment of nanofibers to be deposited into a filter cake layer by layer;
s2, performing suction filtration until the volume of a filter cake is reduced to 1/50 of the volume of the initial filtrate, stopping the suction filtration, rapidly transferring a filter membrane and covering the filter membrane with a new hydrophobic PVDF membrane, placing the filter membrane between water-absorbing papers, compressing and dehydrating the water-absorbing papers under the pressure of 88mbar until the water-absorbing papers are dried, and replacing the water-absorbing papers every 3 hours until the filter membrane is separated from the PVDF membrane, thereby obtaining the TOCNFs film;
s3, immersing the TOCNFs film prepared in the step S2 into a D-PBS solution containing urease with the concentration of 5vol% for 12 hours at the temperature of 4 ℃ to make the film wet and suck the urease into a nanocellulose hydrogel network, so as to realize the construction of an anisotropic hydrogel lamellar structure induced by the wet and swelling of the film;
s4, washing the nano cellulose stent loaded with the urease in the step S3 by deionized water to remove free urease, and immersing the hydrogel into urea-CaCl 2 Mixed solution (comprising urea solution and CaCl with volume ratio of 1:1 and molar concentration ratio of 1:1) 2 The solution is prepared), soaking for 12 hours at 25 ℃, and then rinsing with deionized water for three times to complete the in-situ biomineralization process.
Mineralization process conditions, including urease concentration (10U/ml, 30U/ml, 50U/ml and 70U/ml) in D-PBS solution containing urease, mineralization solution concentration (0.1 mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.9mol/L and 1.1 mol/L), were controlled, and mechanical properties of the scaffolds were monitored, as shown in FIG. 2.
As shown in figure 2, the Young's modulus of the stent under different reaction conditions shows a trend of increasing and then decreasing, and the Young's modulus of the stent under different conditions has anisotropy in the transverse direction (I) and the axial direction (A), and the urease concentration and the mineralization liquid concentration can realize that the mechanical property of the stent is controllable within the range of 10-1000 kPa; in addition, it can be seen from this figure that the higher the concentration of urease, the higher the Young's modulus and the higher the anisotropy of Young's modulus at the same mineralization concentration.
Example 3
S1, diluting 50mg of TOCNFs (total organic carbon nanotubes) by dry weight to 0.1wt% by deionized water, and then carrying out suction filtration to form a membrane by using a hydrophobic PVDF membrane with the pore diameter of 0.22 mu m (Millipore) and the diameter of 47mm under the suction filtration condition of 10mbar of vacuum degree, thereby inducing the alignment of nanofibers to be deposited into a filter cake layer by layer;
s2, performing suction filtration until the volume of a filter cake is reduced to 1/50 of the volume of the initial filtrate, stopping the suction filtration, rapidly transferring a filter membrane and covering the filter membrane with a new hydrophobic PVDF membrane, placing the filter membrane between water-absorbing papers, compressing and dehydrating the water-absorbing papers under the pressure of 88mbar until the water-absorbing papers are dried, and replacing the water-absorbing papers every 3 hours until the filter membrane is separated from the PVDF membrane, thereby obtaining the TOCNFs film;
s3, immersing the TOCNFs film prepared in the step S2 into a D-PBS solution containing urease with the concentration of 5vol% for 12 hours at the temperature of 4 ℃ to make the film wet and suck the urease into a nanocellulose hydrogel network, so as to realize the construction of an anisotropic hydrogel lamellar structure induced by the wet and swelling of the film;
s4, washing the nano cellulose stent loaded with the urease in the step S3 by deionized water to remove free urease, and immersing the hydrogel into 0.5mol/L urea-Ca Cl 2 Soaking the mixture in the mixed solution for 12 hours at 25 ℃, and then rinsing the mixture with deionized water for three times to complete the in-situ biomineralization process.
By regulating urea-CaCl 2 Urea solution and CaCl in the mixed solution 2 Molar concentration ratios of solutions (0.25:1, 0.5:1, 0.75:1, 1:1, 1:0.75, 1:0.5, 1:0.25), caCO of the TOCNFs scaffolds in step 4 were analyzed by TGA 3 The results of the deposition amount of (2) are shown in FIG. 3.
As shown in fig. 3, the precipitated calcium carbonate content at various molar ratios, the TG a analysis gave the need to be found at a molar ratio of 1: caCO can be obtained under the condition 1 3 Is the highest deposition amount of (a).
Example 4
S1, diluting TOBC or TOCNFs with dry weight of 50mg to 0.1wt% by deionized water, and then carrying out suction filtration to form a membrane by using a hydrophobic PVDF membrane with a pore diameter of 0.22 mu m (Millipore) and a diameter of 47mm under the suction filtration condition with a vacuum degree of 10mbar, thereby inducing the orientation arrangement of nanofibers to be deposited into a filter cake layer by layer;
s2, performing suction filtration until the volume of a filter cake is reduced to 1/50 of the volume of the initial filtrate, stopping the suction filtration, rapidly transferring a filter membrane and covering the filter membrane with a new hydrophobic PVDF membrane, placing the filter membrane between water-absorbing papers, compressing and dehydrating the water-absorbing papers under the pressure of 88mbar until the water-absorbing papers are dried, and replacing the water-absorbing papers every 3 hours until the filter membrane is separated from the PVDF membrane, thereby obtaining TOBC and TOCNFs films;
s3, immersing the TOBC and TOCNFs film prepared in the step S2 into a D-PBS solution containing urease with the concentration of 5vol% for 12 hours at the temperature of 4 ℃ to make the film wet and suck the urease into a nanocellulose hydrogel network to realize the anisotropic hydrogel lamellar structure construction induced by the film wet, wherein the concentration of the urease in the D-PB S solution containing the urease is 30U/ml;
s4, washing the nano cellulose bracket loaded with the urease in the step S3 by deionized water to remove free urease, immersing the hydrogel into a urea-CaCl 2 mixed solution with the volume ratio of 1:1 and the molar concentration ratio of 7:4, soaking for 12 hours at 25 ℃, and then rinsing with deionized water for three times to complete the in-situ biomineralization process, wherein the concentration of the urea-CaCl 2 mixed solution is 0.3mol/L;
and (3) placing the in-situ biomineralization anisotropic hydrogel scaffold prepared in the step (S4) and the contrast unmineralization scaffold in a 12-hole plate, and performing ultraviolet irradiation for 30 minutes for sterilization. 2mL of complete medium (containing DMEM, fetal bovine serum, penicillin-streptomycin, L-glutamine) and 1mL of mouse osteosarcoma cell K7M2 with a cell concentration of 10 were added to each well 5 Each mL was placed in a carbon dioxide incubator (37 ℃,5% CO) 2 95% humidity) and measuring the cell viability of mineralized scaffold material and its control unmineralized scaffold by MTT method on days 1,3,5 of culture to evaluate mineralized scaffold cell compatibilityThe results are shown in FIG. 4, where E 0 The urease concentration in the D-PBS solution containing urease was shown, and in addition, cross-sectional images of the C-TOBC scaffolds and C-TOCNFs scaffolds were observed by scanning electron microscopy.
As shown in fig. 4, with the cell viability on the first day TOBC scaffold being 100%, it was observed that the cell number increased continuously with the increase of the culture time, and the cell viability increased. On day 3, cell viability on mineralized scaffolds C-TOBC was significantly greater than the remaining 3 scaffolds (p <0.001 for scaffolds TOBC <0.001 for C-TOCNFs, TOCNFs scaffold p < 0.01). By day 5 of culture, cell viability on the C-TOBC scaffold was significantly greater than on day 3 (p < 0.001), while cell viability on the C-TOBC scaffold was significantly greater than on the remaining 3 scaffolds (p < 0.001). From the figure, the cell viability on the C-TOBC scaffold was significantly better than that of the other 3 groups, which may be related to the mechanical properties of the scaffold being comparable to the mechanical strength of the extracellular matrix required for growth of K7M2 mouse osteosarcoma cells. As shown in FIG. 5, the C-TOBC scaffolds and C-TO CNFs scaffolds were observed by scanning electron microscopy TO exhibit anisotropically oriented structures.
Example 5
Steps S1 to S4 of this example are the same as those of example 2, and mineralized TOBC (H-C-TOBC, 209.5+ -26.3 kPa and L-C-TOBC, 31.6+ -4.3 kPa) with high and low Young' S modulus and a control TOBC holder are selected;
and (3) placing the in-situ biomineralization anisotropic hydrogel scaffold prepared in the step (S4) in a 12-hole plate, and sterilizing by ultraviolet irradiation for 30 minutes. 2mL of complete medium (containing DMEM, fetal bovine serum, penicillin-streptomycin, L-glutamine) and 1mL of mouse mesenchymal stem cells BMSC were added per well at a cell concentration of 5 x 10 3 Each mL was placed in a carbon dioxide incubator (37 ℃,5% CO) 2 95% humidity). ALP kit was used to examine alkaline phosphatase activity of cells on scaffolds of days 1,7 and 14 to judge bone matrix synthesis and mineralization, and the results are shown in FIG. 6.
After the bone tissue repair material is implanted into the body, stem cells are adhered to the scaffold to differentiate into osteoblasts, wherein the mechanical strength of the scaffold material has an important influence on the induced differentiation of cells, so that the effect of the mechanical property of the scaffold on the induced differentiation of cells needs to be studied. Alkaline phosphatase (ALP) is an early marker of osteoblast differentiation, and appears at the beginning of the bone matrix synthesis phase, with a peak mineralization phase. The extent of stem cell differentiation was determined by measuring ALP activity after days 1,7 and 14 of culture, and the results are shown in FIG. 6, in which the intracellular ALP content showed a very significant increase (p < 0.001) with the increase of culture time, indicating that stem cells were induced to differentiate on all three scaffolds. On day 7 of culture, the intracellular ALP content on both mineralized scaffolds was greater than that of TOBC scaffolds and had very significant statistical differences (p < 0.001). On day 14 of culture, the intracellular ALP content on mineralized scaffolds was also extremely significant (p < 0.001) compared to TOBC scaffolds. On the 14 th day of culture, the ALP content in the cells on the H-C-TOBC scaffold is obviously greater than that of the L-C-TOBC scaffold, which indicates that the scaffold with higher mechanical strength can better promote the differentiation of the mesenchymal stem cells BMSC of the mice into osteoblasts, and the differentiation degree of the stem cells can be indirectly regulated by regulating the mechanical property of the mineralized scaffold.
The foregoing description is only a preferred embodiment of the present application, and the present application is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present application has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
1. The preparation method of the in-situ biomineralization-enhancing nanocellulose scaffold is characterized by comprising the following steps:
s1, diluting nano cellulose with deionized water, and carrying out suction filtration on the nano cellulose diluted solution with a hydrophobic PVDF film under a vacuum condition to form a film, thereby inducing the alignment of nano fibers to be deposited layer by layer to form a filter cake;
s2, rapidly transferring a filter cake, covering the filter cake with a new hydrophobic PVDF film, placing the filter cake between water absorbing papers, compressing and dehydrating the filter cake until the filter cake is dried, and replacing the water absorbing papers once every 3 hours until the filter film is separated from the PVDF film, so that the nanocellulose film can be obtained;
s3, immersing the nano cellulose film prepared in the step S2 into a swelling liquid containing urease to swell the film and sucking the urease into a nano cellulose hydrogel network to realize the construction of an anisotropic hydrogel lamellar structure induced by swelling of the film;
s4, washing the nano cellulose bracket loaded with the urease in the step S3 by deionized water to remove free urease, and immersing the hydrogel into mineralized liquid to complete the in-situ biomineralization process.
2. The method for preparing an in situ biomineralization-enhancing nanocellulose scaffold as claimed in claim 1 wherein in step S1 nanocellulose is TOBC or TOCNF; wherein the TOBC has a diameter of 10-20nm, a length of 500-1000nm and a charge density of 1.0mmol/g; the TOCNF has a diameter of 5-10nm, a length of 200-500nm and a charge density of 1.1mmol/g.
3. The method for preparing an in situ biomineralization-enhancing nanocellulose scaffold as claimed in claim 2 wherein in step S1, 50mg dry weight of TOBC or TOCNFs is diluted to 0.1wt% with deionized water, followed by suction filtration to form a membrane with a hydrophobic PVDF membrane with a pore size of 0.22 μm and a diameter of 47mm under suction filtration conditions at a vacuum of 10 mbar.
4. The method for preparing an in situ biomineralization-enhancing nanocellulose scaffold as claimed in claim 1 wherein in step S2, the pressure of compression dehydration is 88mbar.
5. The method for preparing an in-situ biomineralization enhancing nanocellulose scaffold as claimed in claim 1 wherein in step S3, the nanocellulose film is immersed in a swelling solution containing urease at 4 degrees celsius for 12 hours, wherein the concentration of urease in the swelling solution is 10U/mL.
6. The method for preparing an in-situ biomineralization reinforcing nanocellulose scaffold as claimed in claim 1 or 5 wherein said swelling solution is D-PBS and the concentration of said swelling solution is 0 to 100vol%.
7. The method for preparing an in-situ biomineralization-enhancing nanocellulose scaffold as claimed in claim 1 wherein in step S4, the mineralizing solution is urea-CaCl 2 Mixed solution, urea-CaCl 2 The volume ratio of the mixed solution is 1:1 and a molar ratio of urea solution to CaCl of 0.25:1 to 1:0.25 2 The solution is prepared.
8. The method for preparing an in situ biomineralization enhancing nanocellulose scaffold as claimed in claim 1 or 7 wherein the concentration of mineralization solution is 0.1mol/L to 1.1mol/L.
9. The nanocellulose scaffold prepared based on the preparation method of claim 1, wherein the nanocellulose scaffold is in a layered orientation structure.
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