An adjustable alveolar bone repair material and a preparation method thereof

Abstract

The present application relates to a kind of adjustable alveolar bone repair material and its preparation method, comprising the following steps: pig cancellous bone is obtained by pretreatment pig bone powder;Extracellular matrix is mixed with silk fibroin peptide, and extracellular matrix modified by silk fibroin peptide is obtained;Pig bone powder and extracellular matrix modified by silk fibroin peptide are mixed to be uniform, freeze forming is then carried out, and then drying and disinfection are carried out, to obtain the adjustable alveolar bone repair material.The present application has low cost, has good pore structure that helps cell growth and promotes bone, good mechanical properties, does not have cytotoxicity, good antibacterial performance, and has the ability of adjustable, can be cut, can better adapt to bone defect shape.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a tunable alveolar bone repair material and its preparation method. Background Technology

[0002] Tooth defects are a common orthopedic condition in clinical practice. Alveolar bone growth is slow, and the recovery period is long, easily causing serious impact on patients' daily lives, and it remains one of the major challenges in clinical practice. Oral bone restoration typically involves bone grafting or filling with regenerative materials, but the application of autologous bone grafting is limited by the size of the donor site and the defect location. To overcome the shortcomings and limitations of bone grafting, the most common method currently is to use artificial graft materials to replace traditional bone materials, such as framework materials. The complexity and diversity of the oral environment necessitate bone restoration materials with good water absorption and defect adaptability. On the one hand, post-extraction bleeding is a common complication after tooth extraction; if not properly managed, it can lead to wound deterioration and slow down the bone repair rate. On the other hand, the complexity of the oral environment results in a long alveolar bone defect repair period, and current bone framework materials lack good biocompatibility and bioactivity, leading to low osteogenic efficiency.

[0003] Calcined xenogeneic bone is a bone graft material with a natural bone structure. It has abundant raw material sources, an excellent porous three-dimensional structure suitable for new bone growth, good biocompatibility and osteoinductive properties, and poses no harm to the human body after degradation. Bone powder is obtained by grinding calcined xenogeneic bone into particles with a controllable particle size range. Currently, bone powder preparation mostly uses cattle and sheep as raw materials, which is expensive. Although calcined bone powder has many advantages, the powdered material is inconvenient to use, and the problem of difficulties in clinical application of bone powder urgently needs to be solved. Furthermore, for dental patients, bone powder alone cannot effectively solve the problem of poor alveolar bone repair results. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a tunable alveolar bone repair material and its preparation method, thereby solving the technical problems of high cost and poor repair effect of existing alveolar bone repair materials.

[0005] To achieve the above-mentioned technical objectives, the technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing a modifiable alveolar bone repair material, comprising the following steps: pretreating porcine cancellous bone to obtain porcine bone powder; mixing decellularized extracellular matrix with silk fibroin peptides to obtain silk fibroin peptide-modified decellularized extracellular matrix; mixing porcine bone powder and silk fibroin peptide-modified decellularized extracellular matrix until homogeneous, freezing and shaping, and then drying and sterilizing to obtain a modifiable alveolar bone repair material.

[0007] Secondly, the present invention provides a tunable alveolar bone repair material prepared by the above-described preparation method.

[0008] Compared with the prior art, the beneficial effects of the present invention include:

[0009] The alveolar bone repair material prepared by this invention is derived from pigs, resulting in low cost. It utilizes pig bone powder that promotes bone regeneration and silk fibroin peptide-modified extracellular matrix bone scaffold. Tests show that it has a good porous structure that promotes cell growth and bone development, good mechanical properties, no cytotoxicity, good antibacterial properties, and is adaptable and cuttable, which can better adapt to the shape of bone defects. It is a product with very broad application prospects. Attached Figure Description

[0010] Figure 1 These are scanning electron microscope (SEM) images of the alveolar bone repair materials obtained in Examples 1-5 of this invention; where A: 200 μm; B: 20 ​​μm; (a) Example 1 (1g + 0.2g); (b) Example 2 (1g + 0.3g); (c) Example 3 (1g + 0.4g); (d) Example 4 (1g + 0.5g); (e) Example 5 (1g + 0.6g);

[0011] Figure 2 This is an elemental mapping diagram of the alveolar bone repair materials obtained in Examples 1-5 of the present invention, wherein (a) 1g+0.2g; (b) 1g+0.3g; (c) 1g+0.4g; (d) 1g+0.5g; (e) 1g+0.6g;

[0012] Figure 3 These are EDS energy dispersive spectroscopy (EDS) analysis diagrams of the alveolar bone repair materials obtained in Examples 1-5 of this invention, wherein (a) 1g+0.2g; (b) 1g+0.3g; (c) 1g+0.4g; (d) 1g+0.5g; and (e) 1g+0.6g.

[0013] Figure 4 These are FTIR images of the alveolar bone repair materials obtained in Examples 1-5 of this invention;

[0014] Figure 5 These are mechanical property test diagrams of the alveolar bone repair materials obtained in Examples 1-5 of this invention;

[0015] Figure 6 This is an experimental diagram illustrating the antibacterial properties of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] This invention provides an inexpensive, readily available, and malleable alveolar bone repair material by using bone powder that promotes bone regeneration to blend with an extracellular matrix bone scaffold. This material can be blended (by controlling the amount of bone powder and other substances added to improve the scaffold's performance). This invention addresses the problems of high price and difficulty in handling commercially available bovine bone powder, and provides an industrially scalable preparation process to solve the problem of high price of current alveolar bone repair materials.

[0018] Decellularized extracellular matrix (DECM) comprises biomolecules, glycosaminoglycans, proteoglycans, collagen, cell adhesion factors, and growth factors, providing a suitable physiological microenvironment for cell growth, differentiation, and proliferation. Compared to synthetic materials, DECM exhibits high biocompatibility and can accelerate bone regeneration without disrupting the original physiological environment, thus it has been widely applied in the field of bone regeneration. Tissue-derived DECM retains most of its 3D structure and protein components, capturing biophysical and biochemical cues present in natural tissues under physiological conditions. Using DECM as a scaffold can effectively prevent bone powder from being lost due to immersion and erosion by periodontal tissue fluids in the early stages of filling, thereby improving alveolar bone repair efficiency.

[0019] However, direct compounding of bone powder with decellularized extracellular matrix still cannot adapt to the shape of alveolar bone defects, and its clinical application remains difficult.

[0020] Silk fibroin peptides are low-molecular-weight hydrolysates of silk fibroin. They retain the original controlled biodegradability, excellent biocompatibility, and low immunogenicity of silk fibroin, and their small molecular weight makes them more suitable for practical applications. The silk fibroin molecular chain is rich in hydrophilic groups such as -OH, -NH2, and -COOH. These groups not only give SF materials excellent water absorption properties but also allow them to bind Ca through electrostatic interactions and coordination bonds. 2+ It promotes blood clotting, thereby maintaining a favorable physiological microenvironment and accelerating bone regeneration.

[0021] The inventors discovered that combining bone powder with a decellularized extracellular matrix scaffold modified with silk fibroin peptides improves the osteoinductive properties of the scaffold; the silk fibroin peptides provide good water absorption, maintaining a favorable body fluid environment; and the extracellular matrix serves as a medium between the two materials, resulting in a bone scaffold material that can maintain a favorable body fluid environment and has high plasticity. This material can fully simulate the bone tissue microenvironment, alleviating the reduced bone repair efficiency caused by bleeding or a moist oral environment in alveolar bone repair, and solving the problem of inconvenient practical application of bone powder materials.

[0022] This invention provides a composite bone scaffold for alveolar bone defect repair, which uses bone powder made from pigs as raw material. The raw material is widely available and the preparation method is simple, which greatly reduces the cost compared to other methods.

[0023] The present invention discloses a method for preparing a tunable alveolar bone repair material, comprising the following steps:

[0024] (1) Processing of pig bone meal (BP): Pig bone meal is obtained by pre-processing pig cancellous bone.

[0025] (2) Treatment of silk fibroin peptide-modified decellularized extracellular matrix (SP-DECM):

[0026] The decellularized extracellular matrix was mixed with silk fibroin peptides to obtain silk fibroin peptide-modified decellularized extracellular matrix (SP-DECM), which was then stored at 5°C for later use.

[0027] (3) Mix pig bone powder (BP) with silk fibroin peptide modified decellularized extracellular matrix (SP-DECM) until uniform, place in a mold and freeze, then dry and sterilize to obtain a mixable alveolar bone repair material, package for later use.

[0028] Preferably, step (1) specifically includes: purchasing pork bones from a slaughterhouse, cutting off excess parts, retaining the cancellous bone, and cutting it into uniformly sized bone pieces. The bone pieces are laid flat and washed with high-pressure water until no obvious grease seeps out. The washed bone pieces are placed in a pressure cooker at 3-5 atmospheres and cooked for 0.5-1.5 hours. After removal, they are washed with physiological saline and then placed in a solution of 0.2-0.8% (w / w) sodium dodecyl sulfate and 0.5-1.5% (w / w) Triton's solution and stirred overnight. The bone pieces are then washed clean with physiological saline and soaked in 30% hydrogen peroxide and anhydrous ethanol overnight. After removal, they are washed with physiological saline, which is closer to human body fluid, and dried. They are then calcined in a high-temperature furnace at 700-900℃ for 1.5-2.5 hours, cooled to room temperature, pulverized, and sieved (particle size 0.355-0.8 mm) for later use.

[0029] Preferably, the preparation of decellularized extracellular matrix in step (2) specifically includes the following steps: pig Achilles tendons purchased from slaughterhouses are cleaned of excess fat and fascia and cut into small segments to obtain segmented Achilles tendons. These segments are placed in a solution of 0.2-0.8% (w / w) sodium dodecyl sulfate and 0.5-1.5% (w / w) Triton and stirred for 2-4 days. They are then washed with physiological saline, frozen overnight, thawed, and frozen again overnight, repeated 2-4 times. Finally, after thawing, they are placed in 1-3% (w / w) glacial acetic acid and stirred for 2-4 days. They are then taken out and washed 3 times with deionized water, added to a blender, and deionized water is added at a mass ratio of segmented Achilles tendon to water of 1:(2-4) to break them down until there are no large particles, thus obtaining decellularized extracellular matrix.

[0030] Preferably, in step (2), the mass ratio of decellularized extracellular matrix to silk fibroin peptide is (7-9):5.

[0031] Preferably, in step (3), the mass ratio of pig bone powder (BP) to silk fibroin peptide-modified decellularized extracellular matrix (SP-DECM) is (0.2-0.6):1.

[0032] Preferably, in step (3), freezing is performed at -4°C or below for 24 to 72 hours.

[0033] Preferably, in step (3), the drying is freeze drying for 24 to 72 hours.

[0034] The main advantages of this invention are:

[0035] 1. In view of the problem of high cost of existing alveolar bone repair materials, the raw materials of bone powder and decellularized extracellular matrix of this invention are both derived from pigs. In my country, pigs are more widely available and cheaper than cattle, which greatly reduces the cost of bone scaffold.

[0036] 2. In view of the problem of poor water absorption of existing alveolar bone repair materials, the present invention incorporates silk fibroin peptides into the decellularized extracellular matrix, which greatly improves the water absorption of the scaffold material and makes it more effective in surgical procedures.

[0037] 3. To address the issues of small particle size and inconvenient clinical operation of alveolar bone repair materials, this invention combines silk fibroin peptide-modified extracellular matrix and pig bone powder to create a product that is adaptable and cuttable, with greater adaptability to alveolar bone defect shapes and easier clinical application.

[0038] 4. In response to the common problem that alveolar bone repair materials generally lack antibacterial effects, this invention increases the antibacterial properties of the product by introducing silk fibroin peptides. By controlling the ratio of decellularized extracellular matrix to silk fibroin peptides, the antibacterial effect is optimized, and it also facilitates the subsequent addition of bone powder, making it more promising for clinical applications.

[0039] The present invention will be further described in detail below through specific embodiments and comparative examples; to avoid redundancy, the porcine bone meal (BP), decellularized extracellular matrix and SP-DECM used in the following embodiments will be described here.

[0040] Preparation of pork bone meal: Pork leg bones purchased from slaughterhouses were trimmed of excess material, retaining only the cancellous bone, and cut into uniformly sized pieces. The bone pieces were laid flat and washed with a high-pressure water jet until no significant grease exudation was observed. The washed bone pieces were placed in a pressure cooker at 4 atmospheres and boiled for one hour. After removal, they were washed with physiological saline and then placed in a solution of 0.5% (w / w) sodium dodecyl sulfate and 1% Triton's solution, stirred overnight. The bone pieces were then washed again with physiological saline and soaked in 30% hydrogen peroxide and anhydrous ethanol overnight, respectively. After removal, they were washed with physiological saline and dried. The mixture was then calcined in a high-temperature furnace at 800 degrees Celsius for 2 hours, cooled to room temperature, and pulverized to obtain pork bone meal (BP) for later use.

[0041] Preparation of decellularized extracellular matrix: Pig Achilles tendons purchased from slaughterhouses were cleaned of excess fat and fascia, cut into small segments, and placed in a solution of 0.5% (w / w) sodium dodecyl sulfate and 1% Triton and stirred for 3 days. After washing with physiological saline, the tendons were frozen overnight, thawed, and then frozen again overnight, repeated 3 times. Finally, after thawing, the tendons were placed in 2% glacial acetic acid and stirred for 3 days. After washing with deionized water 3 times, the tendons were added to a high-speed blender and deionized water was added at a mass ratio of Achilles tendon to water of 1:3 to break down the tendons until no large particles were present, thus obtaining the decellularized extracellular matrix.

[0042] Since both excessive and insufficient addition of silk fibroin peptides can affect the structure and mechanical properties of the decellularized extracellular matrix scaffold, excessive addition can easily cause the decellularized extracellular matrix structure to become loose, thus affecting the subsequent bone powder composite. Insufficient addition will affect the excellent properties of the scaffold composite silk fibroin peptides. The optimal ratio of 8:5 was selected (i.e., the decellularized extracellular matrix and silk fibroin peptides were mixed at a mass ratio of 8:5 to prepare SP-DECM and stored at 5℃ for later use).

[0043] The silk fibroin peptides were purchased from Shandong Longbei Biotechnology Co., Ltd.

[0044] Example 1:

[0045] Take 0.2g of BP and 1g of (SP-DECM) and mix them until uniform. Place them in a mold and freeze for 48 hours. Take them out and freeze-dry for 48 hours to obtain a white, round, porous solid bone scaffold. Sterilize with ultraviolet light for 2 hours and then package for later use.

[0046] Example 2:

[0047] Take 0.3g of BP and 1g of (SP-DECM) and mix them until uniform. Place them in a mold and freeze for 48 hours. Take them out and freeze-dry for 48 hours to obtain a white, round, porous solid bone scaffold. Sterilize with ultraviolet light for 2 hours and then package for later use.

[0048] Example 3:

[0049] Take 0.4g of BP and 1g of (SP-DECM) and mix them until uniform. Place them in a mold and freeze for 48 hours. Take them out and freeze-dry for 48 hours to obtain a white, round, porous solid bone scaffold. Sterilize with ultraviolet light for 2 hours and then package for later use.

[0050] Example 4:

[0051] Take 0.5g of BP and 1g of (SP-DECM) and mix them until uniform. Place them in a mold and freeze for 48 hours. Take them out and freeze-dry for 48 hours to obtain a white, round, porous solid bone scaffold. Sterilize with ultraviolet light for 2 hours and then package for later use.

[0052] Example 5:

[0053] Take 0.6g of BP and 1g of (SP-DECM) and mix them until uniform. Place them in a mold and freeze for 48 hours. Take them out and freeze-dry for 48 hours to obtain a white, round, porous solid bone scaffold. Sterilize with ultraviolet light for 2 hours and then package for later use.

[0054] Comparative Example 1

[0055] The only difference from Example 4 is that the silk fibroin peptides are replaced with silk fibroin (SF), while the other steps and conditions are the same as in Example 4.

[0056] Performance testing:

[0057] 1. Scanning electron microscope

[0058] Figure 1 The results showed that the products obtained in Examples 1-5 of this invention have a natural three-dimensional porous structure. The pore size of the products ranges from 150 to 500 μm, with pores close to 150 μm being suitable for the inward migration of osteoblasts and inducing bone tissue growth; macropores larger than 300 μm have high permeability and angiogenesis potential, which are beneficial for promoting bone growth and angiogenesis. Figure A shows the successful conjugation of bone powder onto the extracellular matrix scaffold.

[0059] 2. Element mapping

[0060] Figure 2 The results showed that the products obtained in Examples 1-5 of this invention had uniform element distribution and dense structure. The proportion of Ca gradually increased with the increase of bone meal addition, which also indicated the successful compounding of bone meal.

[0061] 3. EDS energy dispersive spectroscopy analysis

[0062] Figure 3 The results show that the minimum Ca:P of the products obtained in Examples 1-5 of this invention is 2.08 > 1.667, which is much greater than the required value of national standard GB23101.3-2010 / ISO13779-3:2008, and meets the national standard.

[0063] 4. FTIR detection

[0064] Figure 4 The results showed that the products obtained in Examples 1-5 of this invention had characteristic peaks at 3575, 1046, and 570, respectively, indicating -OH stretching and PO4 phase expansion. 3- Characteristic peaks and the OPO band indicate that the main component of calcined bone meal is hydroxyapatite. Furthermore, the products obtained in Examples 1-5 show no significant differences. The peak values ​​are similar to those of commercially available products from abroad.

[0065] 5. Mechanical property testing

[0066] A universal tensile testing machine was used to test the compressive properties of the materials. The diameter and height of Examples 1-5 (approximately 15×15mm) were precisely measured using vernier calipers. The compressive strength of each group of materials was determined on the universal testing machine at a loading speed of 10mm / min.

[0067] Figure 5 The results showed that the compressive strength of the products obtained in Examples 1-5 of the present invention all reached the compressive strength of 5-8 MPa of human non-load-bearing bone, meeting the requirements of bone repair materials. Among them, the optimal compressive strength was achieved when the amount of bone powder added in Example 4 was 0.5 g. Too high or too low bone powder content would have a certain impact on the mechanical properties of the material. At the same time, the products obtained in Examples 1-5 of the present invention are all white porous solid bone scaffolds, which can be cut as needed.

[0068] 6. Antibacterial test

[0069] Using the products obtained in Examples 1-5 and Comparative Example 1 (1g + 0.5g (SF)) as test samples, a co-culture experiment was conducted on *Staphylococcus aureus* and *Escherichia coli*. The experimental conditions were as follows: the two strains (*Staphylococcus aureus* and *Escherichia coli*) were taken from the refrigerator and activated in a constant temperature water bath at 37°C, then separately added to liquid culture medium and incubated for 24 hours, and stored for later use. 0.05g of the test sample was weighed and sterilized using ultraviolet light. The sterilized sample was placed in 4mL of liquid culture medium containing bacteria and incubated for 24 hours. 40μL of the bacterial suspension was diluted 10... 5 After treatment, the sample was spread onto a solid culture medium and then incubated on the spread solid culture medium for 24 hours.

[0070] A control group was set up: the liquid culture medium containing bacterial solution was used as a control and cultured under the same conditions for 24 hours, then diluted and spread.

[0071] Depend on Figure 6 It can be seen that the products of this invention all exhibit good antibacterial effects and effectively inhibit bacterial growth after the addition of different amounts of bone powder, indicating that the addition of bone powder has no adverse effect on the antibacterial effect of the composite material. Compared with the products doped with silk fibroin peptides in Examples 1-5, the product doped with silk fibroin in Comparative Example 1 has poor antibacterial effect.

[0072] The control group showed obvious bacterial colonies, which were significantly different from those in Examples 1-5 and more than those in Comparative Example 1.

[0073] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a tunable alveolar bone repair material, characterized in that, Includes the following steps: Pork cancellous bone was pretreated to obtain pork bone meal; The decellularized extracellular matrix was mixed with silk fibroin peptides to obtain silk fibroin peptide-modified decellularized extracellular matrix; the mass ratio of decellularized extracellular matrix to silk fibroin peptides was (7-9):

5. Pig bone powder and silk fibroin peptide-modified decellularized extracellular matrix were mixed until homogeneous, frozen into shape, and then dried and sterilized to obtain a homogeneous alveolar bone repair material; the mass ratio of pig bone powder to silk fibroin peptide-modified decellularized extracellular matrix was (0.2-0.6):

1. The preparation of the decellularized extracellular matrix specifically includes: cutting a segment of porcine Achilles tendon and placing it in a solution of 0.2-0.8% sodium dodecyl sulfate and 0.5-1.5% Triton and stirring for 2-4 days; removing it, washing it with physiological saline, freezing it, and then thawing it, repeating the freeze-thaw cycle 2-4 times; finally thawing it and placing it in 1-3% glacial acetic acid and stirring for 2-4 days; removing it, washing it with deionized water, adding 2-4 times the mass of deionized water, and breaking it down to obtain the decellularized extracellular matrix.

2. The method for preparing the adjustable alveolar bone repair material according to claim 1, characterized in that, The pretreatment of the porcine cancellous bone includes: first, cutting the porcine cancellous bone into uniform bone blocks, washing it with water, and then boiling it for 0.5 to 1.5 hours at 3 to 5 atmospheres; then removing it, washing it, and placing it in a solution of 0.2 to 0.8% sodium dodecyl sulfate and 0.5 to 1.5% Triton overnight; then washing it again and soaking it in 30% hydrogen peroxide and anhydrous ethanol overnight; finally removing it, washing it, drying it, calcining it, cooling it, and then pulverizing it to obtain porcine bone powder.

3. The method for preparing the adjustable alveolar bone repair material according to claim 2, characterized in that, The cancellous bone of the pig was obtained from fresh pig bones; the cleaning was carried out using physiological saline.

4. The method for preparing the adjustable alveolar bone repair material according to claim 2, characterized in that, The calcination is carried out at 700-900℃ for 1.5-2.5 hours.

5. The method for preparing the adjustable alveolar bone repair material according to claim 1, characterized in that, The freeze-forming process involves freezing at -4°C for 24–72 hours.

6. The method for preparing the adjustable alveolar bone repair material according to claim 1, characterized in that, The drying process involves freeze drying for 24–72 hours.

7. A tunable alveolar bone repair material prepared by the preparation method according to any one of claims 1-6.

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