Application of keratin in preparation of bone repair material

Abstract

The invention discloses application of keratin in preparation of a bone repair material, and relates to the technical field of biomedical engineering. The preparation method comprises the following steps: extracting bone marrow mesenchymal stem cells, and carrying out osteogenic induction culture under the intervention of keratin with the concentration of 100-800 [mu] g / ml; the culture conditions of the BMSCs are as follows: in a constant-temperature incubator with the temperature of 37 DEG C and 5% CO2, a culture medium contains a precooled complete culture medium; the first liquid changing time of the primary culture of the BMSCs is 48-72 hours, then the liquid is changed once every 2-3 days, and passage is carried out when the cell fusion degree reaches 80%-90%. Keratin is adopted as a natural biological material, has good biocompatibility and biodegradability and can well interact with cells, and immunological rejection is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to the application of keratin in the preparation of bone repair materials. Background Technology

[0002] Bone defects are a common and complex clinical condition, often caused by trauma, infection, tumor resection, etc. The repair process involves complex biological mechanisms, especially the dynamic balance between osteogenic and osteoclast-reactive processes.

[0003] Bone marrow mesenchymal stem cells (BMSCs) are considered one of the most ideal seed cells for bone tissue engineering due to their multipotent differentiation potential, ease of in vitro expansion, and low immunogenicity. Their osteogenic differentiation capacity can be regulated through physical stimulation, chemical inducers, and biomaterials.

[0004] Currently, while autologous bone transplantation is considered the "gold standard," its sources are limited and donor site complications are numerous; allogeneic bone transplantation carries risks of immune rejection and disease transmission. Therefore, developing bone repair materials with good biocompatibility and osteogenic induction capabilities has become an important research direction in the field of tissue engineering. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of keratin in the preparation of bone repair materials.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The application of keratin in the preparation of bone repair materials, the preparation method includes the following steps: Bone marrow mesenchymal stem cells (BMSCs) were extracted and cultured for osteogenic induction with keratin intervention, the concentration of keratin being between 100 μg / ml and 800 μg / ml.

[0007] Preferably, the BMSCs are cultured in a constant temperature incubator at 37°C and 5% CO2, and the culture medium contains pre-cooled complete culture medium.

[0008] Preferably, the primary culture medium of the BMSCs is changed for the first time at 48-72 hours, and then changed every 2-3 days. When the cell confluence reaches 80%-90%, the cells are passaged.

[0009] Preferably, the passage ratio of the BMSCs is 1:2 to 1:3, and cells from passages 2 to 4 (P2-P4) are selected. The effect of keratin on BMSC cell viability is evaluated by CCK-8 assay, and the specific steps are as follows: Cell suspensions treated with different concentrations of keratin were seeded into 96-well plates, CCK-8 reagent was added, and the OD value of each well was measured after incubation.

[0010] Preferably, in the BMSCs culture, the formation of mineralized nodules is observed and quantitatively analyzed by alizarin red staining. The specific steps are as follows: BMSCs were seeded into 12-well plates and induced to grow for 14-21 days using osteogenic induction medium. After fixation and washing, alizarin red staining was performed, and images were observed and acquired. The size of the red area was quantitatively analyzed.

[0011] Preferably, during the BMSCs culture, the expression levels of OCN and RUNX2 genes are quantitatively analyzed by qPCR, with the following specific steps: Total RNA was extracted, and cDNA was synthesized by reverse transcription. qPCR was then performed on the OCN and RUNX2 genes to analyze changes in gene expression levels.

[0012] Preferably, the preparation method further includes combining keratin with a scaffold material to perform surface modification or coating treatment on the prepared bone repair material.

[0013] Preferably, the keratin is treated at a temperature between room temperature and 37°C in the preparation method, and the treatment time is at least 14 days.

[0014] Preferably, in the preparation method, the purity of BMSCs is higher than 90%.

[0015] The beneficial effects of this invention are as follows: 1. This invention uses keratin, a natural biomaterial, which has good biocompatibility and biodegradability, can interact well with cells, and reduce immune rejection.

[0016] 2. This invention provides a new method to replace autologous bone transplantation or allogeneic bone transplantation, overcoming the risks of limited sources, multiple donor site complications, and immune rejection that exist in traditional methods.

[0017] 3. This invention, through various experimental methods such as CCK-8 assay, alizarin red staining, and qPCR quantitative analysis, has confirmed that keratin treatment does not affect cell viability and can effectively promote osteogenic differentiation, providing strong support for clinical translation.

[0018] 4. The experimental results of this invention show that keratin can significantly upregulate the expression levels of osteogenic-related gene markers OCN and RUNX2, effectively promoting the differentiation of BMSCs into osteoblasts.

[0019] 5. Through experiments involving intervention with different concentrations of keratin, this invention found that the number of mineralized nodules in the high-concentration keratin group increased significantly in a dose-dependent manner, indicating that keratin plays a positive role in regulating osteogenic signaling pathways. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the CCK-8 experimental results of the present invention; Figure 2 This is a schematic diagram of the results of the alizarin red staining experiment of the present invention; Figure 3 This is a schematic diagram of OCN expression levels in the qPCR analysis of this invention; Figure 4 This is a schematic diagram of RUNX2 expression levels in the qPCR analysis of this invention; Figure 5 This is a schematic diagram of alizarin red staining in the BMSC group of this invention; Figure 6 This is a schematic diagram of alizarin red staining in the BMSC-OD group of this invention; Figure 7 This is a schematic diagram of alizarin red staining in the 100 μg / ml BMSC-OD+K group of this invention; Figure 8 This is a schematic diagram of alizarin red staining in the 800 μg / ml BMSC-OD+K group of this invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0022] Example 1: Application of keratin in the preparation of bone repair materials, including.

[0023] I. Extraction and culture of rat BMSCs Euthanasia and disinfection: Rats were euthanized by CO2 asphyxiation or cervical dislocation. The entire surface of the rat was thoroughly disinfected by immersing it in 75% alcohol for 5-10 minutes, and then transferred to a clean bench for aseptic processing.

[0024] Separating the long bones of the lower limbs: Using sterile instruments, sequentially cut open the skin of the back and hind limbs, bluntly dissect the muscle tissue, and fully expose and free the femurs and tibias on both sides. Use scissors and forceps to remove as much muscle, fascia, and connective tissue attached to the bones as possible until the bone surface is smooth. Maintain the integrity of the bones during the procedure to avoid breakage. Immediately place the cleaned long bones into a culture dish containing pre-cooled PBS (containing antibiotics), keep them moist, and wash away any blood.

[0025] Bone marrow extraction: Using sterile scissors, cut off both ends of the long bone (epithelial ends) to fully expose the medullary cavity. Using a 1mL syringe (with a needle of approximately 25G), draw up pre-cooled complete culture medium, insert the needle into one end of the medullary cavity, and slowly inject the medium into the cavity, flushing the bone marrow into a new sterile culture dish. Repeat several times until the medullary cavity turns white. Using the same syringe, repeatedly pipette the bone marrow mass to create a homogeneous single-cell suspension.

[0026] Filtration and resuspension: The cell suspension was filtered through a 70 μm cell sieve into a 50 mL centrifuge tube to remove bone debris and tissue fragments. The culture dish and cell sieve were rinsed with an appropriate amount of PBS and collected into the centrifuge tube.

[0027] Centrifugation and inoculation: After balancing the centrifuge tubes, centrifuge at 1000-1200 rpm (approximately 250-300 × g) for 5 minutes. Discard the supernatant and resuspend the cell pellet in an appropriate amount of pre-warmed complete culture medium. After cell counting, inoculate at (1-2) × 10⁻⁶ cells / mL. 6 cells / cm 2 The cells are seeded at a density in culture flasks or dishes. The culture flasks are labeled (e.g., date, cell type, passage number P0) and incubated in a 37°C, 5% CO2 incubator.

[0028] Primary culture (P0): First medium change: Perform the first complete medium change 48-72 hours after inoculation. At this time, a large number of hematopoietic suspension cells will die or fail to adhere; changing the medium will remove these, preserving adherent BMSCs. Subsequent medium changes: Change the medium every 2-3 days thereafter. When the cell confluence reaches 80%-90%, passage can be performed.

[0029] Subculturing: Discard the old culture medium and gently wash the cell surface 1-2 times with PBS. Add an appropriate amount of 0.25% Trypsin-EDTA and digest at room temperature or 37°C. Observe under a microscope; when the cells become rounded and the gaps between them increase, immediately add an equal volume of complete culture medium to stop the digestion. Gently pipette the bottom of the flask to completely detach the cells, forming a single-cell suspension, and collect it in a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in fresh complete culture medium, and subculture at a ratio of 1:2 to 1:3.

[0030] Experimental passages: Cells from passages 2 to 4 (P2-P4) are generally used in experiments. At this passage level, the cells have high purity, good activity, stable function, and no obvious senescence.

[0031] II. Experimental Grouping BMSCs: Rat bone marrow mesenchymal stem cells BMSC-OD: Osteogenic induction culture using rat bone marrow mesenchymal stem cells BMSC-OD+K (100ug / ml): Low concentration of keratin intervention added to the osteogenic induction culture of rat bone marrow mesenchymal stem cells. BMSC-OD+K (800ug / ml): High concentration of keratin intervention was added to the osteogenic induction culture of rat bone marrow mesenchymal stem cells.

[0032] III. Experimental Methods: 1. Cck-8 experiment: Preparation of cell suspension: When digesting adherent cells, it is recommended to digest them thoroughly and avoid vigorous pipetting to prevent mechanical damage, in order to ensure the accuracy of viable cell counting. After counting, resuspend the cells in complete culture medium to the optimal density determined in the preliminary experiment.

[0033] Plating and culture: Seed the cell suspension into 96-well plates, 100 μL per well. Set up control wells (cells + culture medium + CCK-8) and experimental wells (cells + keratin-containing culture medium + CCK-8). Incubate the plates at 37°C in a 5% CO2 incubator.

[0034] Add CCK-8 reagent: At the planned time point (e.g., 48h), add 10 μL of CCK-8 reagent directly to each well. Gently shake the culture plate to mix.

[0035] Incubation and detection: The culture plate was returned to the incubator to continue incubation for the optimal time determined in the preliminary experiment. After incubation, the OD value of each well was immediately measured using a microplate reader at a wavelength of 450 nm.

[0036] 2. Alizarin Red staining BMSCs were seeded in 12-well plates and induced for 14-21 days using osteogenic induction medium (experimental group containing keratin vs. control group using conventional induction medium), with the medium changed regularly during the period.

[0037] Fixation: Discard the culture medium and gently wash the cells twice with PBS. Add 4% paraformaldehyde or 10% formaldehyde solution to each well and fix at room temperature for 15-30 minutes.

[0038] Washing: Discard the fixative and gently rinse the cells 2-3 times with PBS or deionized water to thoroughly remove the fixative.

[0039] Staining: Add sufficient alizarin red S staining working solution (pH 4.1-4.3) to each well, ensuring complete coverage of the sample, and incubate at room temperature in the dark for 20-30 minutes.

[0040] Cleaning and observation: Carefully aspirate the staining solution and gently rinse several times with deionized water until the eluent becomes clear, avoiding non-specific background staining. Observe and acquire images under an optical microscope; orange-red / dark red mineralized nodules are positive areas.

[0041] Quantitative analysis (optional): ImageJ analyzes the area size of the red region and calculates the percentage for semi-quantitative analysis.

[0042] 3. Quantitative analysis of osteogenic-related gene markers OCN and RUNX2 using qPCR 1. Total RNA Extraction: Discard the culture medium and wash cells twice with PBS. Add TRIzol reagent directly to the wells, lyse the cells, and mix thoroughly by pipetting. Incubate at room temperature for 5 minutes. Following the reagent instructions, add chloroform, isopropanol, and 75% ethanol sequentially for extraction, precipitation, and washing. Finally, dissolve the RNA precipitate in RNase-free water and determine its concentration and purity (A260 / A280 ratio should be between 1.8 and 2.0).

[0043] 2. Reverse transcription (cDNA synthesis): Using a commercial reverse transcription kit, take 1 μg of total RNA as a template, add Oligo(dT) primers, dNTPs, reverse transcriptase, and RNase inhibitor, and perform reverse transcription according to the programmed sequence (usually 25℃ for 5 min, 42℃ for 30-60 min, and 85℃ for 5 min). The obtained cDNA can be diluted for subsequent experiments or stored at -20℃.

[0044] 3. qPCR reaction Primer design: Specific primers were designed for the target gene (Runx2, OCN) and the internal reference gene (β-actin).

[0045] Preparation of the reaction system (taking a 20 μL system as an example): SYBR Green Premix: 10 μL; Upstream primer (10 μM): 0.4 μL; Downstream primer (10 μM): 0.4 μL; cDNA template: 2 μL; ddH2O: 7.2 μL.

[0046] Reaction procedure: Pre-denaturation: 95℃, 30 sec cyclic reaction (40 cycles): 95℃ 5 sec → 60℃ 30 sec (fluorescence signal is collected in this step) Melting curve analysis: 65℃ to 95℃, signal is collected every 0.5℃.

[0047] 4. Data Analysis: The experiment was repeated at least three times. The relative expression levels of genes were calculated using the 2^(-ΔΔCt) method.

[0048] ΔCt = Ct(target gene) - Ct(internal reference gene) ΔCt = ΔCt(experimental group) - ΔCt(control group) Calculate 2^(-ΔΔCt) to obtain the fold change in gene expression in the experimental group relative to the control group.

[0049] IV. Experimental Results: 1. CCK-8 assay confirmed that treatment with keratin at various concentrations did not affect cell viability and demonstrated good biocompatibility.

[0050] 2. Alizarin Red staining experiments confirmed that, compared with the BMSC group, the number of mineralized nodules in the BMSC-OD group increased, indicating effective osteogenic induction. Compared with the BMSC-OD group, the number of mineralized nodules in the BMSC-OD+K (100 μg / ml) and BMSC-OD+K (800 μg / ml) groups was significantly increased in a dose-dependent manner. 3. qPCR analysis showed that the expression levels of OCN and RUNX2 genes were significantly upregulated, indicating that keratin can effectively promote osteogenic differentiation.

[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of keratin in the preparation of bone repair materials, characterized in that, The preparation method includes the following steps: BMSCs were extracted and cultured for osteogenic induction under keratin intervention. The concentration of keratin was between 100 μg / ml and 800 μg / ml, and the purity of BMSCs was higher than 90%.

2. The application of keratin in the preparation of bone repair materials according to claim 1, characterized in that, The BMSCs were cultured in a constant temperature incubator at 37°C and 5% CO2, and the culture medium contained pre-cooled complete culture medium.

3. The application of keratin in the preparation of bone repair materials according to claim 1, characterized in that, The primary culture of BMSCs was first changed after 48-72 hours, and then changed every 2-3 days. When the cell confluence reached 80%-90%, the cells were passaged.

4. The application of keratin according to claim 1 in the preparation of bone repair materials, characterized in that, The BMSCs were passaged at a ratio of 1:2 to 1:3, and cells from passages 2 to 4 were selected. The effect of keratin on BMSC cell viability was evaluated using a CCK-8 assay. The specific steps are as follows: Cell suspensions treated with different concentrations of keratin were seeded into 96-well plates, CCK-8 reagent was added, and the OD value of each well was measured after incubation.

5. The application of keratin according to claim 1 in the preparation of bone repair materials, characterized in that, During the culture of BMSCs, the formation of mineralized nodules was observed and quantitatively analyzed by alizarin red staining. The specific steps are as follows: BMSCs were seeded in 12-well plates and induced to grow on osteogenic induction medium for 14-21 days. After fixation and washing, alizarin red staining was performed, and images were observed and acquired. The size of the red area was quantitatively analyzed.

6. The application of keratin according to claim 1 in the preparation of bone repair materials, characterized in that, During the BMSCs culture, the expression levels of OCN and RUNX2 genes were quantitatively analyzed by qPCR. The specific steps are as follows: Total RNA was extracted, and cDNA was synthesized by reverse transcription. qPCR was then performed on the OCN and RUNX2 genes to analyze changes in gene expression levels.

7. The application of keratin according to claim 1 in the preparation of bone repair materials, characterized in that, The preparation method also includes combining keratin with a scaffold material.

8. The application of keratin according to claim 7 in the preparation of bone repair materials, characterized in that, The preparation method further includes surface modification or coating treatment of the prepared bone repair material.

9. The application of keratin according to claim 8 in the preparation of bone repair materials, characterized in that, In the preparation method, the keratin is treated at a temperature between room temperature and 37°C for at least 14 days.

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