Bioactive glass / recombinant human collagen sponge scaffold and methods of making the same

Abstract

The application discloses a kind of bioactive glass / recombinant human collagen sponge scaffolds and preparation method thereof.The method first mixes recombinant human collagen, bioactive glass and crosslinking agent glycol glycidyl ether uniformly in water to form a mixed solution, freeze-drying, crosslinking at high temperature, and by repeating freeze-drying and water washing step to remove residual crosslinking agent, to obtain bioactive glass / recombinant human collagen sponge scaffold.The bioactive glass / recombinant human collagen sponge scaffold of the application has good biodegradable stability, bone tissue induced regeneration function, high biological activity, good safety, low biological toxicity, and has wide application prospect in bone tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue repair materials technology, and relates to a bioactive glass / recombinant human collagen sponge scaffold and its preparation method. Background Technology

[0002] Bioactive glass, as a novel bone tissue repair material, has attracted widespread attention in the industry due to its excellent biocompatibility and bioactivity. On the one hand, bioactive glass continuously differentiates and interacts with body fluids to form inorganic hydroxyapatite; on the other hand, the released ions can promote the production of growth factors, cell proliferation, and enhance osteoblast gene expression and bone tissue growth. Bioactive glass is the only artificial biomaterial to date that can form bonds with bone tissue while also connecting with soft tissue. However, due to the mismatch in elastic modulus between bioactive glass and bone tissue, its mechanical properties are insufficient, easily leading to bone repair failure, which greatly limits its application. Since some polymers have good mechanical properties, combining bioactive glass with polymers can solve the problem of mismatch in mechanical properties and elastic modulus, and also meet the requirements of transplantation materials. In recent years, bioactive glass composite materials have been widely developed and applied. Currently, the developed composite materials often only consider the elastic modulus of polymers to simulate bone tissue, making it difficult to achieve the synergistic effect of the bioactive effects of polymers and bioactive glass.

[0003] Collagen is an important component of bone tissue and possesses excellent bioactivity. There are reports of combining bioceramics with animal collagen to prepare bone repair scaffolds. Recombinant human collagen (RHC), as a type of collagen, overcomes the risks of rejection and viral infections associated with animal-derived collagen when used in humans, and its human gene recombination better meets the needs of human tissue repair proteins. Summary of the Invention

[0004] The purpose of this invention is to provide a bioactive glass / recombinant human collagen sponge scaffold and its preparation method. This invention, by controlling the ratio of bioactive glass to recombinant human collagen, produces a bioactive glass / recombinant human collagen sponge scaffold with bone tissue regeneration induction function, strong bone tissue adhesion, high biological activity, good safety, and high strength.

[0005] This invention is achieved through the following technical solution:

[0006] A method for preparing a bioactive glass / recombinant humanized collagen sponge scaffold includes the following steps:

[0007] Step 1: Dissolve the recombinant human collagen in water by stirring, according to a mass ratio of bioactive glass to recombinant human collagen of 0.1:1-0.2:1. Then, add the bioactive glass and the cross-linking agent butylene glycol glycidyl ether (BDDE) in sequence, stir until the mixture is uniform, and adjust the pH to alkaline to obtain a mixed solution. The recombinant human collagen is produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021.

[0008] Step 2: Pour the mixed solution into a mold, freeze it rapidly, and then freeze-dry it to obtain a bioactive glass / recombinant human collagen freeze-dried sponge.

[0009] Step 3: Crosslink and solidify the bioactive glass / recombinant human collagen freeze-dried sponge at 50℃~60℃, wash with water, and repeat the freeze-drying and water washing steps twice to obtain the bioactive glass / recombinant human collagen (BG / CHC) sponge scaffold.

[0010] Preferably, in step 1, the bioactive glass is selected from 45S5 bioactive glass, zinc-containing mesoporous bioactive glass, or cerium-containing mesoporous bioactive glass, and more preferably 45S5 bioactive glass.

[0011] Preferably, in step 1, the concentration of bioactive glass in the mixed solution is 2-4 wt%.

[0012] Preferably, in step 1, the concentration of BDDE in the mixed solution is 0.1-2 wt%.

[0013] Preferably, in step 1, the pH is adjusted to 9-11.

[0014] Preferably, in step 2, the rapid freezing temperature is -75℃ to -80℃, and the rapid freezing time is 4-8 hours; the freeze-drying temperature is -75℃ to -80℃, and the freeze-drying time is more than 48 hours.

[0015] Preferably, in step 3, the cross-linking curing time is 4-8 hours.

[0016] Preferably, in step 3, the washing time is 20-24 hours per cycle.

[0017] This invention utilizes the organic-inorganic composite of bioactive glass and recombinant human collagen, and takes advantage of the abundant amino and carboxyl groups on the recombinant human collagen to covalently crosslink with the epoxy groups on the low-biotoxicity crosslinking agent BDDE, thereby achieving the interconnection between recombinant human collagen segments. In this way, the uniformly dispersed bioactive glass particles are fixed in the collagen network, forming a bioactive glass / recombinant human collagen sponge scaffold with a stable interpenetrating network structure that can promote osteogenic differentiation.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The bioactive glass / recombinant human collagen sponge scaffold of the present invention has good biodegradability and bone tissue induction regeneration function. It has high biological activity, good safety and low biotoxicity, and has good tissue compatibility and excellent bone tissue integration.

[0020] (2) This invention introduces bioactive glass into the recombinant human collagen scaffold and controls the ratio between recombinant human collagen and bioactive glass. On the one hand, it enhances the structural stability of the scaffold and promotes rapid osteogenic repair and can achieve the effect of bonding bone tissue. Furthermore, by using BDDE as a crosslinking agent, the mechanical properties and mixing stability of the scaffold are further improved. Its preparation process is simple and can be mass-produced. Attached Figure Description

[0021] Figure 1 This is a photograph of the bioactive glass / recombinant human collagen sponge scaffold prepared in Example 1.

[0022] Figure 2 The images show the SEM images and EDS spectra of RHC, CHC, and BG / CHC sponge stents, where a is the SEM image of the RHC sponge stent, b is the SEM image of the CHC sponge stent, c is the SEM image of the BG / RHC sponge stent, d is the EDS spectra of the RHC sponge stent, e is the EDS spectra of the CHC sponge stent, and f is the EDS spectra of the BG / CHC sponge stent.

[0023] Figure 3 Tensile strength and elongation at break of RHC, CHC, and BG / CHC sponge scaffolds with different bioactive glass contents are shown.

[0024] Figure 4 The degradation rates of RHC, CHC, and BG / CHC sponge scaffolds with different bioactive glass contents are shown.

[0025] Figure 5 Cell proliferation curves for CHC sponge scaffolds and BG / CHC sponge scaffolds with different bioactive glass contents.

[0026] Figure 6 The expression levels of BMP 2, OPN, β-Actin, and SP7 in CHC sponge scaffolds and BG / CHC sponge scaffolds with different bioactive glass contents. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be purchased commercially.

[0028] The recombinant human collagen used in the following examples and comparative examples was produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021, which has been fully disclosed in Chinese Patent ZL 201110327865.5, and was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0029] Example 1

[0030] Preparation of 2wt% bioactive glass / recombinant human collagen (2wt% BG / CHC) sponge scaffold:

[0031] 2.0 g of recombinant human collagen was added to 7.6 g of water and stirred for 30 min. Then, 0.2 g of bioactive glass was added and stirred for 30 min. Subsequently, 0.2 g of BDDE was added as a cross-linking agent, and the pH was adjusted to 9-10. The mixture was stirred for 30 min to obtain a mixed solution. The mixed solution was poured into a mold and rapidly frozen at -75℃ for 5 h, followed by freeze-drying for 48 h to obtain a lyophilized sponge of bioactive glass / recombinant human collagen. The lyophilized sponge of bioactive glass / recombinant human collagen was placed in a 60℃ forced-air drying oven for 6 h, and finally washed with pure water for 24 h. The freeze-drying and washing steps were repeated twice to obtain a 2 wt% BDDE-crosslinked bioactive glass / recombinant human collagen sponge scaffold.

[0032] Example 2

[0033] Preparation of 4wt% bioactive glass / recombinant human collagen (4wt% BG / CHC) sponge scaffold:

[0034] 2.0 g of recombinant human collagen was added to 7.4 g of water and stirred for 30 min. Then, 0.4 g of bioactive glass was added and stirred for 30 min. Subsequently, 0.2 g of BDDE was added as a cross-linking agent, and the pH was adjusted to 9-10. The mixture was stirred for 30 min to obtain a mixed solution. The mixed solution was poured into a mold and rapidly frozen at -75℃ for 5 h, followed by freeze-drying for 48 h to obtain a lyophilized sponge of bioactive glass / recombinant human collagen. The lyophilized sponge of bioactive glass / recombinant human collagen was placed in a 60℃ forced-air drying oven for 6 h, and finally washed with pure water for 24 h. The freeze-drying and washing steps were repeated twice to obtain a 4 wt% BDDE-crosslinked bioactive glass / recombinant human collagen sponge scaffold.

[0035] Comparative Example 1

[0036] Preparation of BDDE-crosslinked recombinant human collagen sponge (CHC) scaffold:

[0037] 2.0 g of recombinant human collagen was added to 7.8 g of water and stirred for 30 min. Then, 0.2 g of BDDE was added as a cross-linking agent, and the pH was adjusted to 9-10. The mixture was stirred for 30 min to obtain a mixed solution. The mixed solution was poured into a mold and rapidly frozen at -75℃ for 5 h, followed by freeze-drying for 48 h to obtain a recombinant human collagen freeze-dried sponge. The recombinant human collagen freeze-dried sponge was placed in a 60℃ forced-air drying oven for 6 h, and finally washed with pure water for 24 h. The freeze-drying and washing steps were repeated twice to obtain a BDDE-crosslinked recombinant human collagen sponge scaffold.

[0038] Comparative Example 2

[0039] Preparation of recombinant human collagen (RHC) sponge scaffold:

[0040] Add 2.0g of recombinant human collagen to 8.0g of water, stir for 30min, adjust the pH to 9-10, stir for 30min to obtain a collagen solution. Pour the collagen solution into a mold, rapidly freeze at -75℃ for 5h, and then freeze-dry for 48h to obtain a recombinant human collagen sponge scaffold.

[0041] Comparative Example 3

[0042] Preparation of a 6wt% bioactive glass / recombinant human collagen (6wt% BG / CHC) sponge scaffold:

[0043] 2.0 g of recombinant human collagen was added to 7.2 g of water and stirred for 30 min. Then, 0.6 g of bioactive glass was added and stirred for 30 min. Subsequently, 0.2 g of BDDE was added as a cross-linking agent, and the pH was adjusted to 9-10. The mixture was stirred for 30 min to obtain a mixed solution. The mixed solution was poured into a mold and rapidly frozen at -75℃ for 5 h, followed by freeze-drying for 48 h to obtain a lyophilized sponge of bioactive glass / recombinant human collagen. The lyophilized sponge of bioactive glass / recombinant human collagen was placed in a 60℃ forced-air drying oven for 6 h, and finally washed with pure water for 24 h. The freeze-drying and washing steps were repeated twice to obtain a 6 wt% BDDE-crosslinked bioactive glass / recombinant human collagen sponge scaffold.

[0044] Characterization example

[0045] 1. Tensile test

[0046] The mechanical properties of each sponge scaffold were determined according to the test method of pharmaceutical industry standard YY / T 0471.4-2004. The specific steps are as follows: The sponge scaffold samples were cut into 2cm × 8cm pieces and then fixed on a texture analyzer with a clamping distance of 5cm. A uniaxial tensile test was conducted under constant temperature and humidity conditions of 25℃ and 50% to test the tensile strength of the sponge scaffold.

[0047] 2. Degradation rate test

[0048] First, accurately weigh the sponge scaffold and record the initial mass as Wo. Immerse the sponge scaffold in excess PBS aqueous solution and place it in a 37°C incubator for 3 days. After immersion, remove the sponge scaffold, rinse it three times with distilled water, then freeze it overnight, freeze-dry it, and weigh it, recording the weight as Wt. Calculate the degradation rate in water using the following formula: Degradation rate (%) = (Wo - Wt) / Wo × 100%.

[0049] The degradation stability of the sponge scaffold can be judged by the degradation rate; the lower the degree of degradation, the more stable the surface structure.

[0050] 3. Cell proliferation experiment using sponge scaffolds

[0051] The proliferation of BMSCs in sponge scaffolds was detected using the CCK8 assay. BMSCs were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 96-well plates. Cell proliferation was observed on days 1, 3, 5, and 7, with the medium changed every two days. At the end of the experimental period, the medium was replaced with basal medium containing 10% (v / v) CCK8 reagent, and cultured for another 6 hours. The absorbance of 100 μL of supernatant was measured at 450 nm using a Tecan Infinite F200 / M200 multi-plate reader (Tecan, Mannedorf, Switzerland).

[0052] 4. Detection of specific osteogenic gene expression levels in sponge scaffolds

[0053] BMSCs cells were loaded at a rate of 1×10 5Cells were seeded at a density in 24-well plates fixed with sponge scaffolds. After 7 days of culture, the relative expression levels of specific genes (BMP2, ALP, OPN, SP7, β-Actin) in osteoblasts were assessed using real-time quantitative PCR. The cell scaffold complex was washed with PBS, 1 mL of RNAex was added, the culture plate was gently shaken, and cells were repeatedly pipetted to detach them. The supernatant was transferred to EP tubes and incubated at room temperature for 5 min. 200 μL of chloroform was added, the mixture was vortexed, and incubated at room temperature for 5 min. The mixture was then centrifuged at 12000 g, 4 °C for 15 min. The supernatant was carefully transferred to a new centrifuge tube, 500 μL of isopropanol was added, and the mixture was thoroughly mixed and incubated at room temperature for 10 min. The mixture was centrifuged at 12000 g, 4 °C for 10 min, the supernatant was discarded, the precipitate was collected, dried, and dissolved in DEPC-treated water. The RNA concentration was measured. cDNA was obtained by reverse transcription and Real-Time PCR was performed under standard reaction conditions of the Thermal Cycler Dice Real-Time System. After the reaction, confirm the amplification and melting curves of Real-Time PCR. Prepare a standard curve for PCR quantification using 2... ΔΔCt Calculate the expression level of each target gene relative to the previous time point. Verify the amplification efficiency of the primers and quantitatively compare gene expression.

[0054] Figure 1 The image shows a physical example of a bioactive glass / recombinant human collagen sponge scaffold. It can be seen that the bioactive glass / recombinant human collagen sponge scaffold can be processed into different shapes, and the surface texture is uniform.

[0055] Figure 2 The images show SEM and EDS spectra of RHC, CHC, and BG / CHC sponge scaffolds. Figures a and c show that all sponge scaffolds have a relatively uniform porous structure. The scaffold surfaces of RHC and CHC sponge scaffolds are smooth, while Figure c shows particles distributed on the scaffold surface of the BG / CHC sponge scaffold. This indicates that bioactive glass is dispersed and fixed within the collagen sponge scaffold. The EDS analysis in Figure df further confirms that the particles on the surface of the BG / CHC sponge scaffold are bioactive glass.

[0056] Figure 3 The figures show the tensile strength and elongation at break of RHC, CHC, and BG / CHC sponge scaffolds with different bioactive glass contents. As can be seen from the figures, the tensile strength and elongation at break of the sponge scaffold first increase and then decrease with increasing bioactive glass content. This is because while the addition of bioactive glass enhances the scaffold strength, excessive addition leads to excessive overall rigidity of the material, indicating that a high content of inorganic particles is detrimental to the elongation at break.

[0057] Figure 4 The degradation rates of RHC, CHC, and BG / CHC sponge scaffolds with different bioactive glass contents are shown. The degradation rates of each sponge scaffold on days 3, 7, 14, 21, and 28 are displayed. It can be seen that the degradation rate is significantly reduced after BDDE cross-linking and decreases further with the increase of bioactive glass content. The results indicate that BDDE covalent cross-linking and the addition of bioactive glass effectively improve the degradation stability of collagen sponge scaffolds.

[0058] Figure 5 The figures show cell proliferation curves for CHC sponge scaffolds and BG / CHC sponge scaffolds with different bioactive glass contents. As can be seen from the figures, the sponge scaffold with 2 wt% BG / CHC showed a significantly higher induced proliferation effect than the CHC sponge scaffold, indicating that the addition of bioactive glass synergistically induces proliferation with CHC. Conversely, the 6 wt% BG / CHC sponge scaffold showed a detrimental effect on cell proliferation, suggesting that the ratio of bioactive glass to recombinant human collagen in the sponge scaffold has a significant impact on cell compatibility.

[0059] Figure 6 The expression levels of four osteogenic-related genes—BMP2, OPN, βActin, and SP7—were observed in the CHC sponge scaffold and in BG / CHC sponge scaffolds with different bioactive glass contents. It can be seen that at 7 days, the expression levels of BMP2 in the BG / CHC sponge scaffold were 2.3-fold and 1.7-fold higher than those in the NC and CHC sponge scaffolds, respectively; OPN was 1.5-fold and 1.5-fold higher; βActin was 2-fold and 1.3-fold higher; and SP7 was 2.0-fold and 1.8-fold higher. These results indicate that the BG / CHC sponge scaffold can effectively promote osteogenic differentiation.

Claims

1. A method for preparing a bioactive glass / recombinant human collagen sponge scaffold, characterized in that, Includes the following steps: Step 1: Dissolve recombinant human collagen in water by stirring, using a bioactive glass to recombinant human collagen mass ratio of 0.1:1-0.2:

1. Then, add the bioactive glass and cross-linking agent BDDE sequentially, stirring until homogeneous. Adjust the pH to 9-11 to obtain a mixed solution. The recombinant human collagen is produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021. In the mixed solution, the concentration of bioactive glass is 2-4 wt%, and the concentration of BDDE is 0.1-2 wt%. Step 2: Pour the mixed solution into a mold, rapidly freeze and then freeze-dry to obtain a bioactive glass / recombinant human collagen freeze-dried sponge. The rapid freezing temperature is -80℃ to -75℃ and the rapid freezing time is 4-8 hours. The freeze-drying temperature is -80℃ to -75℃ and the freeze-drying time is more than 48 hours. Step 3: Cross-link and cure the bioactive glass / recombinant human collagen freeze-dried sponge at 50℃~60℃ for 4-8 hours. Wash with water and repeat the freeze-drying and water washing steps twice to obtain the bioactive glass / recombinant human collagen sponge scaffold.

2. The preparation method according to claim 1, characterized in that, In step 1, the bioactive glass is selected from 45S5 bioactive glass, zinc-containing mesoporous bioactive glass, or cerium-containing mesoporous bioactive glass.

3. The preparation method according to claim 1, characterized in that, In step 3, the washing time is 20-24 hours per cycle.

4. The bioactive glass / recombinant human collagen sponge scaffold prepared by any one of the preparation methods according to claims 1 to 3.

5. The application of the bioactive glass / recombinant human collagen sponge scaffold according to claim 4 in the preparation of bone tissue repair materials.

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