CN110787322A - A kind of mineralized keratin biomimetic material and preparation method thereof - Google Patents

Abstract

The invention discloses a preparation method of a mineralized keratin biomimetic material, which comprises the following operation steps: 1) adding nano-scale keratin powder at a concentration of 0.1-500 mg/ml into a sterile simulated body fluid environment, and allowing it to stand for mineralization treatment , and then collected, vacuum freeze-dried, and ground into powder to obtain mineralized keratin particles; 2) The mineralized keratin particles were resuspended at a concentration of 0.1-500 mg/ml and injected into the template, and then placed in an oven to dry, A film-like mineralized keratin biomimetic material was obtained. The invention induces the self-mineralization of keratin through the biomimetic synthesis method, and constructs the biomimetic material of mineralized keratin without introducing other protein factors or other biological macromolecules. Practice shows that the biomimetic material can provide an ideal nano-mineralized microenvironment for the growth and proliferation of DPSCs to promote the growth of DPSCs, thus providing an important reference value for the feasibility of pulp tissue regeneration and repair.

Description

A kind of mineralized keratin biomimetic material and preparation method thereof

technical field

The invention relates to biomedical materials, in particular to a mineralized keratin biomimetic material.

Background technique

Pulp inflammation, pulp necrosis and various kinds of pulp diseases are common and high-incidence oral diseases. The pulp tissue necrosis and severe inflammatory reaction accompanying the disease process will lead to severe pain, which is very important to the patient's normal diet and life. cause adverse effects. At present, root canal filling is the main treatment method for this type of disease. Because the disease has caused damage to the tissues and cells inside the root canal of the tooth, the treatment idea is to kill or remove the normal and diseased tissues in the pulp cavity. The root canal is filled with materials or drugs to relieve pain, reduce inflammation and prevent the spread of lesions. Materials for root canal filling can be divided into three types: solid, liquid and paste according to their physical properties. Among them, solid fillings are often dominated by Malay latex, metallic silver or polypropylene. These materials are used in treatment. There are pros and cons to each process. However, the fundamental role of the current root canal treatment is to use inactive materials to eliminate the root canal and pulp cavity, eliminate the dead space, eliminate the source of re-infection, and does not repair the pulp, and the loss of the pulp will lead to the loss of the hard tissue of the tooth. To the source of nutrition, the fragility increases, and the incidence of tooth fracture is significantly higher than that of living pulp teeth. Therefore, the increasingly perfect root canal treatment plan in the future should be a strategy that can promote pulp regeneration and build healthy teeth. Therefore, finding a material that can promote pulp tissue regeneration is one of the hot spots and difficulties in pulp regeneration research.

From the perspective of tissue structure, the tissue structure of dental pulp in the root canal is relatively simple compared with the tissue structure in other organs: the root canal is hollow and contains dental pulp stem cells (DPSCs); Peripheral differentiation to form odontoblasts and ameloblasts; peripheral nerves and blood vessels enter the pulp through the apical foramen at the bottom of the root to nourish and regulate the tooth. Current studies have shown that the environment in which DPSCs live will affect their growth, proliferation, and differentiation. In particular, the external environment of calcification may interact and regulate each other with various cells in the dental pulp, especially DPSCs. On the one hand, the ossification, odontoblastization or odontoblastization of DPSCs will increase the calcium content in the extracellular environment, and the final formation of this calcium environment is the purpose of pulp, enamel and dentin repair On the other hand, the calcium ion environment improves the bioactivity and biocompatibility of the material interface, promotes the proliferation and differentiation of DPSCs, and can significantly increase the number of the latter: for example, Tu et al. found that compared with blank In terms of group, the cell viability of DPSCs treated with hydroxyapatite was higher; Chen et al.'s study showed that the mineralized surface of the material would improve the osseointegration ability of DPSCs, make it better integrate with the material interface, and grow more Tight cellular structure; Türkkan et al. found that an increase in the CaP concentration in the medium would facilitate the adhesion and growth of DPSCs to the material.

Therefore, whether to create an ideal mineralized microenvironment for DPSCs to promote their proliferation and differentiation into odontoblasts is a key to the regeneration of dental pulp tissue. Biomimicry research provides ideas for finding and constructing such ideal materials. Building materials under the conditions of simulating the internal environment of organisms is one of the current research methods in the field of biomaterials. It is worth mentioning that the ultimate purpose of biomimetic synthesis of various biological materials is by no means simply replicating biological minerals, but guiding the synthesis of artificial mineralized materials on the basis of understanding the principles of biomineralization in nature. The synthesis method of materials eventually creates various inorganic materials with superior properties. However, mineralization cannot be generated out of thin air and requires biological macromolecules as scaffolds and media, and currently there is no ideal mineralized microenvironment that can effectively and stably promote the proliferation and differentiation of DPSCs into odontoblasts.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a preparation method of a mineralized keratin biomimetic material for the deficiencies of the above-mentioned prior art, and the obtained mineralized keratin biomimetic material will provide an ideal nano-mineralization microenvironment for the growth of DPSCs, And regulate the growth and proliferation of DPSCs, so as to obtain new filling materials for pulp tissue regeneration.

The present invention introduces biomimetic synthesis, which, in short, simulates the process of natural biomineralization, makes the surface of biomacromolecules mineralize and accumulates in simulated body fluids, synthesize tricalcium phosphate, hydroxyapatite or similar structure. In recent years, research reports using biological macromolecules such as proteins and nucleic acids as templates have gradually increased. For example, Xu et al. used this method to construct a mineralized bioglass scaffold to promote the growth of bone cells on it; Li et al. used the phenolic hydroxyl group on L-dopamine to induce the formation of hydroxyapatite, and developed a biological component Similar PDA composite powder; Zhang et al. also used dopamine as a template to synthesize needle-type and microsphere-type hydroxyapatite.

However, mineralization cannot be produced out of thin air, and requires biological macromolecules as scaffolds and media. Among biological macromolecules, keratin is an ideal structural material. The protein monomers are tubular, and two protein monomers are coiled with each other to form structural units, and the structural units are arranged in parallel, forming cross-links with each other by hydrogen bonds, showing an ordered structure in space. Because of its tough properties, keratin is the main component of many biological organs, such as nails, bird beaks or hair. Therefore, keratin is also regarded as one of the ideal materials for constructing biomimetic scaffolds.

Due to the presence of free sulfhydryl groups in cysteine and its low water solubility, keratin has mechanical structural plasticity and can be improved. Therefore, biological structures constructed from keratin often have enhanced mechanical strength. In addition, keratin also possesses cell-binding motifs such as leucine-aspartate-valine (LDV), which may potentially facilitate cell-matrix interactions. At present, research on keratin is focused on the field of evolutionary biology, and researchers in the field of materials science have also begun to try to use keratin as a scaffold. Zhao et al. used keratin to improve the mechanical properties of caprolactone scaffolds and found that the scaffolds containing cross-linked keratin were suitable for supporting cell attachment and proliferation, and the cells on it showed better cell viability than the control group; for another example, Xu et al. Studies have shown that keratin contains leucine-aspartate-valine (LDV) cell adhesion motifs and some regulatory molecules that can enhance nerve tissue regeneration, which is suitable for scaffold modification for skin tissue engineering. s Choice. These studies suggest that keratin may also have good biocompatibility for DPSCs, and at the same time, the scaffold material based on it may also be an ideal controlled-release drug carrier. The present invention conducts an in-depth study based on the above scientific hypothesis, that is, whether an exogenous mineralized microenvironment can positively influence the growth and proliferation of DPSCs.

The technical scheme adopted in the present invention is: a preparation method of a mineralized keratin biomimetic material, which comprises the following operation steps:

1) Nano-level keratin powder is added to a sterile simulated body fluid environment at a concentration of 0.1-500 mg/ml, left to stand for mineralization, and then centrifuged at room temperature, discard the supernatant and collect the precipitate, carry out resuspension cleaning, vacuum freezing Dry and grind into powder to obtain mineralized keratin particles;

2) The mineralized keratin particles obtained in step 1) were resuspended at a concentration of 0.1-500 mg/ml and then injected into the template. After the precipitation was complete, it was placed in an oven at 20-85°C and dried for 1-5 hours to obtain film-like minerals. Keratinized biomimetic material.

In the operation process of the present invention, other protein factors or other biological macromolecules other than keratin as raw materials are not introduced, but only a bionic environment is created for keratin by simulating body fluids, and the process of simulating natural biomineralization allows keratin The protein achieves self-mineralization, and the surface of keratin is mineralized and accumulated to form a biomimetic structure that is conducive to the growth and proliferation of DPSCs.

Wherein, in the present invention, the concentration of keratin in the simulated body fluid in step 1) is limited to be 0.1-500 mg/ml. This is because when the concentration of keratin is too low, that is, less than 0.1 mg/ml, the hydrophilicity and dispersibility in water after mineralization will increase, which will cause the operation defect that the mineralization is not easy to be collected by centrifugation. When the keratin concentration is too high, that is, higher than 500 mg/ml, it tends to agglomerate in simulated body fluids, resulting in insufficient mineralization. In addition, the present invention defines that the concentration of mineralized keratin in the simulated body fluid in step 2) is also 0.1-500 mg/ml. This is because when the mineralized keratin concentration is too high or too low, it is not conducive to stably form a film-like biomimetic material in the template. Therefore, limiting the concentration of keratin in the simulated body fluid in the two steps of the present invention is one of the important process parameters for finally obtaining the film-like biomimetic material. Through further experimental exploration of the present invention, it is preferable to control the concentration of keratin in both steps 1) and 2) to be 50-200 mg/ml.

It is worth mentioning that the template described in step 2) of the present invention is the surface that provides support for the synthesis of the mineralized keratin membrane and is directly defined accordingly. Therefore, in the actual operation of the present invention, a cell culture plate can be selected as the Formwork can also be made of similar PVC material with flat structure formwork container.

As a further improvement of the above scheme, the standing time in step 1) is 1-6 weeks. Specifically, the static mineralization treatment described in step 1) of the present invention is a key operation to change the surface morphology and structure of keratin. After a large number of experimental studies, it was found that when the static mineralization treatment time reached 1 week, the changes in the surface morphology and structure of keratin also began to be prominent with the increase of the static mineralization treatment time; when the static mineralization treatment time increased. At 6 weeks, the change of keratin surface morphology and structure was the most significant, and the subsequent keratin membrane structure was smoother, and the materials were closely connected with each other, showing a biomimetic structure similar to the internal microstructure of dentin. ; When the mineralization treatment time was 6 weeks, the changes of keratin surface morphology and structure gradually tended to stop.

As a further improvement of the above scheme, the centrifugation in step 1) is centrifugation at 1000-15000 rpm for 1-30 min.

As a further improvement of the above scheme, the particle size of the mineralized keratin particles in step 1) is 20-80 nm. Specifically, the particle size of the mineralized keratin particles in step 1) of the present invention gradually decreases with the increase of the standing mineralization treatment time, and when the particle size of the mineralized keratin is 20-80 nm , which is beneficial to the subsequent formation of a biomimetic membrane structure similar to the microstructure inside the dentin.

The beneficial effects of the invention are as follows: the invention induces the self-mineralization of keratin through the biomimetic synthesis method, and constructs the biomimetic material of mineralized keratin without introducing other protein factors or other biological macromolecules. Practice shows that the biomimetic material can provide an ideal nano-mineralized microenvironment for the growth and proliferation of DPSCs to promote the growth of DPSCs, thus providing an important reference value for the feasibility of pulp tissue regeneration and repair.

Description of drawings

Fig. 1 is the quality detection of keratin particles in embodiment 1 and comparative example;

Fig. 2 is the infrared spectrum results of the keratin particles mineralized for 1 week and mineralized for 6 weeks in Comparative Example and Example 1;

Figure 3 is the XPS results of the keratin particles mineralized for 6 weeks in Comparative Example and Example 1;

Figure 4 is the TEM and SAED results of the keratin particles mineralized for 6 weeks in Comparative Example and Example 1 (the left is the transmission electron microscope photo; the right is the selected area electron diffraction pattern);

Figure 5 is the SEM results of keratin particles mineralized for 1 week and mineralized for 6 weeks in Comparative Example and Example 1;

Fig. 6 is the analysis result of keratin particle size in embodiment 1 and comparative example;

Fig. 7 is the SEM (A) and AFM (B) of the keratin film material mineralized for 1 week and mineralized for 6 weeks in Comparative Example and Example 1, wherein the upper layer is the unmineralized keratin of the blank control group; the middle layer is the mineralized keratin Mineralized keratin for 1 week; the lower layer for mineralized keratin for 6 weeks;

Fig. 8 is the surface roughness analysis result of the keratin film material mineralized for 1 week and mineralized for 6 weeks in Comparative Example and Example 1;

Fig. 9 is the surface hydrophilicity analysis result of the keratin membrane material mineralized for 1 week and mineralized for 6 weeks in Comparative Example and Example 1;

Figure 10 is the MTT detection results of DPSCs on the surface of different materials in Example 6 after culturing 4h, 24h, 48h and 72h, wherein "control" refers to the cell control group, "keratin" refers to the blank control group of the comparative example, and "mineralization" refers to the blank control group. "Keratin" refers to the test sample mineralized for 6 weeks in Example 1;

Figure 11 is the flow cytometry detection of the DPSCs on the surface of different materials in Example 6 after culturing for 72 hours, wherein "control group" refers to the cell control group, "keratin" refers to the blank control group of the comparative example, and "mineralized keratin" Refers to the test sample mineralized for 6 weeks in Example 1;

Figure 12 is the SEM test results of DPSCs on the surface of different materials in Example 6 after culturing for 24 and 72 hours, wherein "keratin" refers to the blank control group of the comparative example, and "mineralized keratin" refers to the mineralized keratin in Example 1 for 6 weeks. test sample.

Detailed ways

The present invention will be specifically described below with reference to the embodiments, so as to facilitate the understanding of the present invention by those skilled in the art. It is necessary to point out that the embodiments are only used to further illustrate the present invention, and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art make non-essential improvements and The adjustment should still belong to the protection scope of the present invention. Meanwhile, the raw materials mentioned below are all commercially available products that are not described in detail; the process steps or preparation methods not mentioned in detail are the process steps or preparation methods known to those skilled in the art.

The nanoscale protein powder described in the embodiment of the present invention is the mouse-derived keratin powder purchased from Guangzhou Huaqisheng Biotechnology Co., Ltd.; the sterile simulated body fluid is purchased from Guangzhou Huaqisheng Biotechnology Co., Ltd. The sterile simulated body fluid SBF; the low-glucose αMEM medium was a commercially available product from GIBCOBRL; the newborn bovine serum was a commercially available product from Hangzhou Sijiqing Bioengineering Materials Co., Ltd.

Example 1: Construction of mineralized keratin biomimetic material

A preparation method of a mineralized keratin biomimetic material, comprising the following steps:

1) Get the nanoscale keratin powder and add it to the sterile simulated body fluid environment at a concentration of 100mg/ml, leave it to stand for mineralization for 6 weeks, stir and disperse slightly every 12h, and take samples every 1 week as a sample, when sampling The mineralized keratin was placed in a 1.5ml EP tube, centrifuged at 10000rpm for 15min at room temperature, the supernatant was discarded, the precipitate was collected, resuspended with deionized sterile water and washed 3 times, vacuum freeze-dried, and ground to a particle size of 50nm powder, mineralized keratin particles with mineralization for 1 to 6 weeks were obtained and used for subsequent physical and chemical characterization experiments;

2) Take the mineralized keratin particles obtained in step 1) that have been mineralized for 1 to 6 weeks and resuspend them at a concentration of 100 mg/ml and inject them into a 24-well cell culture plate. Placed in a 50° C. oven to dry for 3 hours, respectively, to obtain film-like mineralized keratin biomimetic materials that were mineralized for 1 to 6 weeks.

Example 2: Construction of mineralized keratin biomimetic material

A preparation method of a mineralized keratin biomimetic material, comprising the following steps:

1) Take nano-scale keratin powder and add it to the sterile simulated body fluid environment at a concentration of 0.5mg/ml, let it stand for mineralization treatment for 6 weeks, stir and disperse slightly every 12h, take samples when the mineralization is over, and place the mineralized keratin In a 1.5ml EP tube, centrifuge at 15,000rpm for 1min at room temperature, discard the supernatant, collect the precipitate, resuspend with deionized sterile water and wash 3 times, vacuum freeze-dry, and grind into a powder with a particle size of 20nm to obtain the mineral. Keratinized particles;

2) The mineralized keratin particles obtained in step 1) were resuspended at a concentration of 500 mg/ml and injected into a 24-well cell culture plate. After they were deposited on the bottom of the 24-well cell culture plate, they were placed in a 20°C oven to dry. 5h, the mineralized keratin biomimetic material of Example 2 was obtained.

Example 3: Construction of mineralized keratin biomimetic material

A preparation method of a mineralized keratin biomimetic material, comprising the following steps:

1) Take the nano-scale keratin powder and add it to the sterile simulated body fluid environment at a concentration of 500mg/ml, leave it to stand for mineralization for 6 weeks, slightly stir and disperse every 12h, take samples when the mineralization is over, and place the mineralized keratin in the environment. In a 1.5ml EP tube, centrifuge at 1000rpm for 30min at room temperature, discard the supernatant, collect the precipitate, resuspend with deionized sterile water and wash 3 times, vacuum freeze-dry, grind into a powder with a particle size of 80nm to obtain mineralization keratin particles;

2) The mineralized keratin particles obtained in step 1) were resuspended at a concentration of 0.1 mg/ml and injected into a 24-well cell culture plate. After they were deposited on the bottom of the 24-well cell culture plate, they were placed in an oven at 85°C. After drying for 1 hour, the mineralized keratin biomimetic material of Example 3 was obtained.

Comparative Example: Constructing a Blank Control

A kind of preparation method of keratin film material, it comprises the following operation steps:

1) Get the nanoscale keratin powder with a concentration of 100mg/ml and add it to the sterile simulated body fluid environment, place it in a 1.5ml EP tube, and centrifuge it at 10000rpm for 15min at room temperature, discard the supernatant, collect the precipitation, and carry out with deionized sterile water Resuspend and wash 3 times, vacuum freeze-dry, grind into powder with particle size of 50nm, and obtain blank control group keratin particles;

2) The keratin particles obtained in step 1) were resuspended at a concentration of 100 mg/ml and injected into a 24-well cell culture plate. The blank control group was keratin film.

Example 5: Physicochemical characterization of mineralized keratin biomimetic material

5.1 Quality inspection of mineralized keratin particles

Take the blank control group keratin particles of the comparative example and the mineralized keratin particles of Example 1 for 1 to 6 weeks for quality testing, respectively, and the test results are shown in Figure 1. The quality of the test samples has increased. Specifically, after 6 weeks of mineralization, the mass of the test sample increased from 0.1 g in the blank control group to 0.143 ± 0.006 g, and the mass growth rate was 14%, which was a significant difference, which means that the keratin after mineralization treatment A new substance is formed on the surface.

5.2 Infrared analysis of mineralized keratin particles

The blank control group keratin particles of the comparative example, the mineralized keratin particles mineralized for 1 week and the mineralized keratin particles for 6 weeks in Example 1 were taken respectively, 2 g each, and the KBr tablet method was used for Fourier transform infrared spectroscopy analysis to control the sample. The particle size of the mixture with KBr is less than 2 μm, so as to avoid the influence of scattered light, and the infrared spectrum detection results are shown in Figure 2. The results showed that mineralization changed the infrared absorption peak of keratin, especially after 6 weeks of mineralization, the change was more significant. For example, according to the detection data of "mineralized keratin 6w" (ie, mineralized keratin particles that have been mineralized for 6 weeks), there are distinct peaks at 1045.2 cm ^-1 and 1174.0 cm ^-1 , which are hydroxyapatite, respectively The vibration characteristic peaks of v ₁ and v ₃ of PO ₄ ^3- in the middle show that the structure of keratin has changed after mineralization, and new chemical bonds are generated, which causes the change of the infrared spectrum of the sample.

5.3 XPS analysis of mineralized keratin particles

Take the blank control group keratin particles of the comparative example and the mineralized keratin particles in Example 1 for 6 weeks respectively, and use a MICROLAB MK II photoelectron spectrometer to measure the XPS energy spectrum of the film of each test sample. For Ca and P, the spatial resolution of the electron gun is 50 nm, and the voltage is 10 kV. The XPS test results are shown in Figure 3. The test results show that after 6 weeks of mineralization of the tested sample in Example 1, obvious characteristic peaks appear at the XPS binding energies of 347eV and 133eV, indicating that the mineralized keratin surface is enriched with more calcium ions and phosphorus ions, Combined with the above analysis results of Fourier transform infrared spectrum, it can be known that the newly formed substances on the surface of keratin after mineralization may be analogs of hydroxyapatite.

5.4 TEM and SAED analysis of mineralized keratin particles

The blank control group of the comparative example and the mineralized keratin particles mineralized for 6 weeks in Example 1 were respectively taken for TEM and SAED analysis. As shown in Figure 4 (left), 6 weeks of mineralization changed the structure of keratin. Specifically, the mineralization increased the granularity of keratin, the electron density of keratin decreased, the boundary became obvious, and the particles were separated from each other. . As shown in Figure 4 (right), the unmineralized keratin SAED in the blank control group did not show obvious crystal regularity, the distances between the bright spots in the image were different, and the points were scattered, but basically arranged around the center of the pattern On the concentric circles, it is the pattern diffracted by the electron beam after passing through the α-helix of keratin; and after 6 weeks of mineralization, regular quadrilateral structures and bright spots appear on the SAED spectrum of the test sample. They are arranged uniformly and are regular quadrilaterals. From this, it is inferred that mineralization can change the surface morphology of keratin, and the crystal structure of tetrahedron appears.

5.5 SEM analysis of mineralized keratin particles

The blank control group keratin granules of the comparative example and the mineralized keratin granules in Example 1 were respectively taken for 6 weeks, and the tested samples were naturally air-dried and then pasted and fixed on the sample table and sprayed with gold. In the scanning electron microscope sample chamber, vacuumize and observe the electron microscope. The observation results are shown in Figure 5 (blank control group in the left picture, mineralized for 1 week in the middle picture, and mineralized for 6 weeks in the right picture). The boundary is not obvious, but after 1 week and 6 weeks of mineralization, the keratin has obvious granular structure, and the particles are uniform. The analysis results corresponded to the TEM detection results.

5.6 Mineralized keratin particle size

Take the keratin particles of the blank control group of the comparative example and the mineralized keratin particles of Example 1 for 1 to 6 weeks for particle size detection respectively. The detection results are shown in Figure 6. It is found that the particle size of keratin increases with The increase of mineralization time showed a gradual downward trend. Specifically, the particle size of keratin particles in the blank control group of the comparative example was 100 nm. After 3 weeks of mineralization, the particle size decreased to 50 nm, a decrease of 50%. The particle size of mineralized keratin particles can reach 30 nm after 6 weeks, and the detection results of 1 to 6 weeks are compared with the blank control group of the comparative example, P<0.01, which is statistically different.

5.7 Surface morphology of mineralized keratin film

Take the blank control group keratin particles of the comparative example, and the mineralized keratin biomimetic materials in Example 1 that were mineralized for 1 week and mineralized for 6 weeks to observe the surface morphology by SEM and AFM, respectively. As shown in Figure 7, the mineralization Different treatment time will have an impact on the effect of keratin film formation. Specifically, the film structure formed by unmineralized keratin in the blank control group has more pore structures and the surface is uneven. In contrast, the mineralization treatment 6 The membrane structure formed by the mineralized keratin after weeks is smoother, the materials are closely connected with each other, and the structure presented is similar to the fine structure inside the dentin. This surface may simulate the mineralized keratin biomimetic material of the present invention. The internal structure of the tooth provides an ideal microenvironment for subsequent growth and differentiation of DPSCs. 5.8 Surface roughness of mineralized keratin film

代表该层平均高度。计算出每一层的Ra后，再计算出同一角蛋白膜材的200个片层的平均Ra值，以此代表其粗糙度。如图8所示，随着矿化时间的延长，所形成的角蛋白膜材的粗糙度逐渐下降，实施例两个受试样品的粗糙度较空白对照组的下降了50％，这与矿化角蛋白颗粒的SEM结果相对应，表明本发明的矿化角蛋白仿生材料表面的空隙减少、表面变得平整。The keratin particles of the blank control group of the comparative example and the mineralized keratin biomimetic materials of Example 1 that were mineralized for 1 week and mineralized for 6 weeks were respectively used for roughness analysis. The specific operation method is to take a 10μm×10μm area on the upper surface of each test sample, divide it into 200 layers for scanning, and each layer is divided into 200 scanning points, so each area is divided into 4×10 ⁴ scan points. The data scanned by the instrument were processed by NanoScope Analysis 1.5 (Bruker, Germany) and OriginPro 9 (OriginLab, USA) to obtain visualized 3D and 2D graphics. Then, the formula of the average roughness Ra of each layer of the keratin film is defined as follows: In the formula, Zn represents the height of each scanning point,

Represents the average height of the layer. After calculating the Ra of each layer, calculate the average Ra value of 200 layers of the same keratin film to represent its roughness. As shown in Figure 8, with the extension of the mineralization time, the roughness of the formed keratin film gradually decreased, and the roughness of the two test samples in Example decreased by 50% compared with that of the blank control group, which was consistent with that of the blank control group. The SEM results of the mineralized keratin particles correspond to each other, indicating that the voids on the surface of the mineralized keratin biomimetic material of the present invention are reduced and the surface becomes smooth.

5.9 Hydrophilicity of mineralized keratin film

Take the blank control group keratin particles of the comparative example, and the mineralized keratin biomimetic materials mineralized for 1 week and mineralized for 6 weeks in Example 1 for surface hydrophilicity analysis respectively, as shown in FIG. The film formed by mineralized keratin has higher hydrophilicity, which is manifested as a decrease in the surface contact angle value, which is significantly higher than that of the blank control group and the test sample mineralized for 1 week in Example 1. On the surface, the mineralized keratin biomimetic material of the present invention has better biocompatibility and is more favorable for the growth of DPSCs thereon.

Example 6: Cell Culture

Rat dental pulp stem cells (DPSCs) provided by Saibaikang (Shanghai) Biotechnology Co., Ltd. were taken and dispensed into 24-well or 96-well cell culture plates, and the keratin membrane materials of the blank control group were added respectively. With the mineralized keratin biomimetic material mineralized for 6 weeks in Example 1, the overall quality of the keratin added in each hole is guaranteed to be consistent, and the pure rat dental pulp without keratin membrane material or mineralized keratin biomimetic material is used. Stem cells served as a cell control group. After the cells were confluent and cultured to 80% in the culture flask, they were seeded on the cell culture plate at a density of 1×10 ⁴ to 3×10 ⁴ /well, and cultured for 72 hours for subsequent MTT detection, flow cytometry and scanning electron microscopy. Observed. Other cell culture conditions were: low glucose αMEM medium containing 10% newborn calf serum, 37°C, 5.0% CO ₂ .

6.1MTT detection

Discard the culture medium from the cell liquid after cell culture, add MTT with a concentration of 5 mg/ml, the ratio is 1:10, and incubate at 37 °C for 4 h. Discard the supernatant, add 100 μL of DMSO, shake sufficiently, dissolve the crystals, detect the absorbance at 490 nm with a microplate reader, and perform MTT detection. The detection results are shown in Figure 10 . According to the MTT detection results, DPSCs showed significantly different growth conditions, and the results of the test samples mineralized for 6 weeks in Example 1 were significantly better than those of the blank control group and the cell control group in the comparative example. From the data point of view, after culturing for 24h, the OD value of the test sample after 6 weeks of mineralization was about 3 times that of the cell control group and 2 times that of the blank control group, respectively, and this fold relationship was observed after 48h and 72h of cell culture. Hours later, it was further expanded. After culturing for 72 hours, the OD value of the test sample that had been mineralized for 6 weeks was ~3.0, while the OD value of the cell control group was less than 1.0, that is, the mineralized keratin biomimetic material of the present invention was more suitable for the growth of DPSCs and could significantly enhance the growth of DPSCs. promote the proliferation of the cells.

6.2 Flow Cytometry

Cells were collected after 72 h of cell culture, and counted by flow cytometry. At the same time, the cell cycle was detected by flow cytometry to characterize the proliferation of cells. The test results are shown in Figure 11. In Example 1, a relatively high proportion of cells on the test sample that had been mineralized for 6 weeks passed the G1 cycle checkpoint, entered the S, G2 or M phase, and were in the G1/G0 phase. Compared with the cell control group, the proportion of cells in the S-phase decreased by 21%, while the proportion of S-phase cells in the test sample that had been mineralized for 6 weeks in Example 1 was higher, at 26.71%, and there was a significant difference compared with the other two groups, while The proportion of cells in G2/M phase in the blank control group was higher, which was 22.23%

6.3 Scanning electron microscope observation

As shown in FIG. 12 , it can be seen that the surface morphology of the keratin membrane material was changed by the cell culture. Comparative example The surface of the keratin film in the blank control group has many undulations, and its local structure mostly presents a spherical or granular structure, and the particle surface is smooth and has no extra structure. The surface of the biomimetic material was flat as a whole, and a tubular structure appeared at the same time. It was inferred that the tubular structure should be the cohesin or collagen grown on the DPSCs that could be used to attach the material.

In conclusion, the mineralized keratin biomimetic material of the present invention has good biocompatibility for DPSCs, and can provide a good environment for cell growth and proliferation, so as to promote cell growth. Therefore, it has the potential to be further developed into biomaterials that can be used for pulp tissue repair.

The above-mentioned embodiments are preferred embodiments of the present invention, and all processes similar to those of the present invention and equivalent changes made shall belong to the protection scope of the present invention.

Claims (6)

1. A preparation method of mineralized keratin bionic material is characterized by comprising the following operation steps:

1) adding the nanoscale keratin powder into a sterile simulated body fluid environment at a concentration of 0.1-500 mg/ml, standing for mineralization, centrifuging at room temperature, discarding the supernatant, collecting the precipitate, carrying out heavy suspension cleaning, vacuum freeze drying, and grinding into powder to obtain mineralized keratin particles;

2) and (2) resuspending the mineralized keratin particles obtained in the step 1) at the concentration of 0.1-500 mg/ml, injecting the resuspended mineralized keratin particles into a template, and drying the template for 1-5 hours in an oven at the temperature of 20-85 ℃ after complete precipitation to obtain the filmy mineralized keratin bionic material.

2. The method for preparing the mineralized keratin biomimetic material according to claim 1, wherein the method comprises the following steps: the standing time in the step 1) is 1-6 weeks.

3. The method for preparing the mineralized keratin biomimetic material according to claim 1, wherein the method comprises the following steps: the centrifugation treatment in the step 1) is centrifugation for 1-30 min under the condition of 1000-1500 rpm.

4. The method for preparing the mineralized keratin biomimetic material according to claim 1, wherein the method comprises the following steps: the particle size of the mineralized keratin particles in the step 1) is 20-80 nm.

5. A mineralized keratin biomimetic material prepared by the preparation method of any one of claims 1 to 4.

6. Use of a mineralized keratin biomimetic material as defined in claim 5 to promote the growth and proliferation of DPSCs.

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