A hydrogel / calcined bone porous composite scaffold for physical assisted bone repair material and its preparation method and application

Abstract

The present invention discloses a hydrogel / calcined bone porous composite scaffold for physical assisted bone repair materials and a preparation method and application thereof, and relates to the technical field of biomaterials. The present invention combines the calcined bone with a hydrogel material containing free ions by ionic crosslinking, and then prepares a calcium phosphate mineralization layer on the surface of the obtained composite material to obtain the hydrogel / calcined bone porous composite scaffold. The hydrogel / calcined bone porous composite scaffold with good biocompatibility and ultrasonic electric field response prepared by the present invention has good bone generation ability, does not need to assemble other circuit elements, has good formability, has the function of constructing a dynamic electric field under ultrasonic response, and has extremely broad application prospects in the fields of physical therapy and bone repair.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a hydrogel / calcined bone porous composite scaffold for physical auxiliary bone repair material, and a preparation method and application thereof. Background Art

[0002] Natural bone has piezoelectric effect, and electrical signals are important physiological signals for bone tissue growth. The lack of physiological electrical signals at the bone defect site is an important factor leading to difficulty in bone repair and poor regeneration quality. Integrating electrical signals in bone repair materials and activating osteogenic signal pathways are effective means to improve the repair effect of bone defect repair materials. Therefore, the development of bone graft materials with electrical signals has become a new way of bone repair.

[0003] Traditional piezoelectric materials, such as inorganic piezoelectric materials based on lead titanate and barium titanate or organic piezoelectric materials based on polyvinylidene fluoride-hexafluoropropylene copolymer (PVDF) and polylactic acid (PLLA), have stable electrical output performance, but are still subject to some application restrictions in bone repair. Among them, materials such as lead titanate, barium titanate, and PVDF are not biodegradable and are not suitable for long-term existence in the body; PLLA has good biodegradability and stable electrical output function, but its price is relatively expensive, and the raw material preparation process is cumbersome.

[0004] At present, researchers have prepared ionic piezoelectric hydrogels with good biocompatibility, which use mechanical force to induce the diffusion difference of anions and cations inside the hydrogel to induce dynamic potential changes and generate voltage signals. Ultrasound (US) is a periodic mechanical wave that is controllable, non-invasive and highly penetrating in human tissue and is widely used in clinical diagnosis. Therefore, using ultrasound as a trigger for ion movement inside the hydrogel is an effective way to output electrical stimulation. Summary of the invention

[0005] The purpose of the present invention is to provide a hydrogel / calcined bone porous composite scaffold for physical assisted bone repair material and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is to provide a method for preparing a hydrogel / calcined bone porous composite scaffold, comprising the following steps:

[0008] The calcined bone is combined with a hydrogel material containing free ions through ion cross-linking, and then a calcium phosphate mineralization layer is prepared on the surface of the obtained composite material to obtain the hydrogel / calcined bone porous composite scaffold.

[0009] Furthermore, the calcium phosphate mineralization layer is prepared by biomineralization.

[0010] Furthermore, the biomineralization method is specifically to soak in dipotassium hydrogen phosphate solution and calcium chloride solution in sequence.

[0011] Furthermore, the calcination temperature of the calcined bone is 800° C. and the calcination time is 4 hours.

[0012] Furthermore, the hydrogel material is sodium alginate hydrogel; and the free ions are sodium ions and chloride ions.

[0013] Furthermore, the ionic crosslinking is carried out in a diffusion-mediated manner; and the solution used for the ionic crosslinking is a calcium chloride solution.

[0014] A more preferred preparation method comprises the following steps:

[0015] (1) dissolving sodium alginate powder in 0.9% physiological saline to obtain a hydrogel prepolymer containing sodium and chloride ions;

[0016] (2) dissolving calcium chloride in deionized water to obtain a calcium chloride solution;

[0017] (3) cutting the bovine cancellous bone into appropriate sizes, calcining it in a muffle furnace (heating from room temperature to 800° C., heating rate of 10° C. / min, keeping warm for 4 h) to remove fat and other components in the bovine bone, thereby obtaining calcined bone;

[0018] (4) mixing the hydrogel prepolymer of step (1) with the calcined bone and placing the mixture in a diffusion mold, and then removing the air in the pores using a vacuum oven so that the hydrogel is fully filled in the calcined bone;

[0019] (5) placing a wet semipermeable membrane on the surface of the mold so that it contacts the hydrogel inside the mold, then docking the same molds (with the same interface size), injecting the calcium chloride solution into the upper layer and letting it stand for a certain period of time to obtain a hydrogel / calcined bone porous material.

[0020] (6) The cross-linked hydrogel / calcined bone porous material is immersed in a K2HPO4·3H2O solution, and then taken out and immersed in a CaCl2·2H2O solution to prepare a calcium phosphate mineralization layer on the surface of the material, thereby obtaining a hydrogel / calcined bone porous composite scaffold.

[0021] In the present invention, the mold used can be prepared by 3D printing technology, PDMS molding technology or other processing technology.

[0022] More preferably, the concentration of the dissolved sodium alginate powder is 0.02-0.05 g / mL, most preferably 0.02 g / mL; the concentration of the calcium chloride solution is 0.1-1 M, most preferably 1 M.

[0023] More preferably, the standing time in step (5) is 3-6 hours; the soaking time in step (6) is 1-5 minutes, most preferably 3 minutes.

[0024] More preferably, the concentration of the K2HPO4·3H2O solution is 100-300 mM, most preferably 300 mM, and the concentration of the CaCl2·2H2O solution is 100-500 mM, most preferably 500 mM.

[0025] The second technical solution of the present invention is to provide a hydrogel / calcined bone porous composite scaffold prepared by the above preparation method.

[0026] The present invention uses high-temperature calcined bone as a matrix, composites a hydrogel containing free ions on the matrix, obtains a porous composite scaffold with ultrasonic electric field response through diffusion-mediated ion cross-linking, and then uses biomineralization technology to cover a layer of calcium phosphate matrix on its surface. Among them, the hydrogel provides space for ion movement; the calcined bone provides a new bone formation framework for bone repair, and the surface mineralized coating can provide more sites for cell adhesion.

[0027] The hydrogel / calcined bone porous composite scaffold obtained by the present invention has the function of constructing a dynamic electric field under ultrasonic response, and has extremely broad application prospects in the fields of physical therapy and bone repair.

[0028] The third technical solution of the present invention is to provide the application of the above-mentioned hydrogel / calcined bone porous composite scaffold as a bone defect filling material.

[0029] The steps for repairing bone defects using the hydrogel / calcined bone porous composite scaffold of the present invention are as follows:

[0030] The gel / calcined bone porous composite scaffold is used to repair the bone defect, and ultrasound is used as an excitation source to achieve the repair of the bone defect.

[0031] Specifically, the frequency of the ultrasonic wave is 1 MHz, the duty cycle is 20%, and the intensity is 10-30 mW / cm 2 .

[0032] The present invention uses non-immunogenic high-temperature calcined bone as the core of the porous composite scaffold for bone repair, combines the free ion-rich hydrogel (power-generating matrix) with the calcined bone material through a diffusion-mediated ionic cross-linking method, and then uses biomineralization technology to prepare a calcium phosphate coating on the material surface that is conducive to cell adhesion. Using the penetrating effect of ultrasound, the free ions in the gel move differentially to generate electrical signals, which further affect cell behavior and enhance the bone repair effect.

[0033] The present invention discloses the following technical effects:

[0034] The present invention combines the calcined bone porous material with the hydrogel containing free ions by means of diffusion-mediated ionic cross-linking, and utilizes the difference in physical properties of sodium ions and chloride ions in the physiological environment to produce differentiated migration under the action of ultrasonic therapy, thereby constructing a dynamic electric field in the repair material and the defect area, thereby improving the bone repair effect.

[0035] The hydrogel / calcined bone porous composite scaffold with good biocompatibility and ultrasonic electric field response prepared by the present invention has good bone formation ability, does not need to assemble other circuit elements, and has good formability.

[0036] The hydrogel / calcined bone porous composite scaffold of the present invention can stably output electrical signals in combination with low-frequency ultrasound, promote cell behavior and provide a good microenvironment suitable for bone defect repair.

[0037] The preparation process of the present invention is simple, the cycle is short, the material source is wide, the price is low, and the preparation process is environmentally friendly and pollution-free, with strong repeatability. The obtained hydrogel / calcined bone porous composite scaffold has stable electrical output under ultrasonic response, and can be mass-produced industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 Surface scanning electron microscope images of the calcined bone porous material, the hydrogel / calcined bone porous material and the hydrogel / calcined bone porous composite scaffold prepared in Example 1 of the present invention.

[0040] Figure 2 This is a cross-sectional scanning electron microscope image of the hydrogel / calcined bone porous material prepared in Example 1 of the present invention.

[0041] Figure 3 This is a graph of the output electrical signals of the hydrogel / calcined bone porous composite scaffold prepared in Example 1 of the present invention under ultrasound action.

[0042] Figure 4 The absorbance of the calcined bone porous material, the hydrogel / calcined bone porous material and the hydrogel / calcined bone porous composite scaffold prepared in Example 1 of the present invention.

[0043] Figure 5 Scanning electron microscope images of the calcined bone porous material, hydrogel / calcined bone porous material and hydrogel / calcined bone porous composite scaffold prepared in Example 1 of the present invention after cell co-culture.

[0044] Figure 6 These are cell images of the control group and material group under ultrasonic and non-ultrasonic conditions in Application Example 5 of the present invention. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] Example 1

[0051] Preparation of a hydrogel / calcined bone porous composite scaffold for physical assisted bone repair material:

[0052] (1) Weigh 0.2 g of sodium alginate powder and mix with 10 mL of 0.9% saline to prepare a sodium alginate solution with a concentration of 0.02 g / mL;

[0053] (2) Weigh 1.11 g of anhydrous calcium chloride and mix thoroughly with 10 mL of deionized water to prepare an anhydrous calcium chloride solution with a concentration of 1 M;

[0054] (3) Weigh 6.85 g of K2HPO4·3H2O and mix thoroughly with 100 mL of deionized water to prepare a 300 mM K2HPO4·3H2O solution;

[0055] (4) Weigh 5.55 g of anhydrous CaCl2 and mix thoroughly with 100 mL of deionized water to prepare a CaCl2 solution with a concentration of 500 mM;

[0056] (5) The bovine cancellous bone was cut into appropriate sizes, and then calcined in a muffle furnace (heating from room temperature to 800°C, heating rate of 10°C / min, and keeping warm for 4 hours) to remove fat and other components in the bovine bone, thereby obtaining a calcined bone porous material (labeled as TBC);

[0057] (6) Mixing the sodium alginate solution obtained in step (1) and the calcined bone porous scaffold obtained in step (5) and placing them in a diffusion mold, and then removing the air in the pores using a vacuum oven so that the hydrogel fully fills the inside of the porous scaffold; placing a wet semipermeable membrane on the surface of the above mold so that it contacts the hydrogel inside the mold, docking the same mold, injecting the above calcium chloride solution into the upper layer and letting it stand for 5 hours to obtain a hydrogel / calcined bone porous material (labeled as TBCH);

[0058] (7) The obtained hydrogel / calcined bone porous material was placed in a K2HPO4·3H2O solution for 3 min, taken out and immersed in a CaCl2·2H2O solution for 3 min, and the cycle was repeated 3 times to obtain a composite scaffold with a calcium phosphate mineralization layer, namely, a hydrogel / calcined bone porous composite scaffold (labeled as CaP@TBCH).

[0059] Application Example 1

[0060] The surface scanning electron microscope images of the calcined bone porous material, hydrogel / calcined bone porous material and hydrogel / calcined bone porous composite scaffold prepared in Example 1 are as follows: Figure 1 As shown in the figure, the calcined bone porous material presents an interconnected porous morphology, and the hydrogel is physically bonded to the surface of the hydrogel / calcined bone porous material. After biomineralization, the surface of the porous composite scaffold is obviously covered with a coral-like calcium phosphate coating (CaP coating).

[0061] In order to prove that the hydrogel was filled into the calcined bone porous material, a scanning electron microscope was used to photograph its cross section ( Figure 2 ).from Figure 2 It can be seen that the interior of the calcined bone porous material is filled with hydrogel, indicating that the hydrogel is fully combined with the calcined bone porous material.

[0062] Application Example 2

[0063] The hydrogel / calcined bone porous composite scaffold prepared in Example 1 was subjected to ultrasound (frequency of 1 MHz, duty cycle of 20%, intensity of 30 mW / cm 2 ) induces the differential migration of internal sodium ions and chloride ions in the dynamic electric field of the internal components of the material. Using an oscilloscope to test, the voltage can reach 80mV ( Figure 3 ), which can trigger cell membrane action potentials and thus effectively have a positive impact on cell behavior.

[0064] Application Example 3

[0065] The calcined bone porous material, hydrogel / calcined bone porous material and hydrogel / calcined bone porous composite scaffold prepared in Example 1 were co-cultured with cells. Mouse mesenchymal stem cells were used as the cell source for co-culture with the three materials. Cell proliferation test (CCK-8) was performed after 7 days of culture. The results are as follows: Figure 4 shown.

[0066] Cell co-culture: First, sterilize the material and soak it in 75% alcohol twice, 20 min each time, wash off the excess alcohol with sterile PBS, and place the sterilized material in a 24-well plate; then collect and resuspend the cells that have grown well after recovery, and place them in a 24-well plate at 2×10 4 / well with a pipette into the corresponding well plate. Each group of materials has three parallel groups. After completing the above operations, the well plate was placed in a cell culture incubator (37°C, 5% CO2), and the medium was changed on the fourth day. CCK-8 detection was performed after 7 days of culture;

[0067] During the cell culture process, the above materials were divided into an ultrasound group (+US) and a non-ultrasound group (-US). The ultrasound had a frequency of 1 MHz, a duty cycle of 20%, and an intensity of 30 mW / cm 2 , ultrasound once a day, each time for 10 minutes.

[0068] Depend on Figure 4 It can be seen that under ultrasound, the hydrogel / calcined bone porous composite scaffold has a higher absorbance, indicating the largest number of surviving cells.

[0069] Application Example 4

[0070] The calcined bone porous material, hydrogel / calcined bone porous material and hydrogel / calcined bone porous composite scaffold prepared in Example 1 were co-cultured with cells. Mouse mesenchymal stem cells were used as the cell source for co-culture with the three materials. After 7 days of culture, the cells were fixed for scanning electron microscopy imaging test. The results are as follows: Figure 5 shown.

[0071] Cell co-culture: First, sterilize the material and soak it in 75% alcohol twice, 20 min each time, wash off the excess alcohol with sterile PBS, and place the sterilized material in a 24-well plate; then collect and resuspend the cells that have grown well after recovery, and place them in a 24-well plate at 2×10 4 Each well was added into the corresponding well plate with a pipette. Each group of materials had three parallel groups. After completing the above operations, the well plate was placed in a cell culture incubator (37° C., 5% CO2), and the medium was changed on the fourth day. CCK-8 detection was performed after 7 days of culture.

[0072] Since the calcined bone porous material is a macroscopic macroporous structure, only a small number of cells adhere to the calcined bone material; for hydrogel / calcined bone porous materials, since the material is already filled with hydrogel, cells can directly adhere to the surface of the hydrogel, and sodium alginate hydrogel is a hydrogel with excellent biocompatibility, so cells can adhere well to the surface of the hydrogel; for hydrogel / calcined bone porous composite scaffold, after mineralization modification, a layer of CaP coating is deposited on the surface, which can release calcium ions and phosphate ions that are beneficial to the activity of mesenchymal stem cells, and the relatively rough surface of the coating is more conducive to cell adhesion, showing a larger number of cells. For bone repair materials, cell recruitment is a prerequisite for repair, and the hydrogel / calcined bone porous composite scaffold of the present invention is a potential bone repair material.

[0073] Application Example 5

[0074] The hydrogel / calcined bone porous composite scaffold prepared in Example 1 was used to repair femoral defects in New Zealand rabbits. A control group (i.e., no material was implanted, 3 New Zealand white rabbits in each group at each time point, about 4 months old) and a material group (i.e., implanted material, 3 New Zealand white rabbits in each group at each time point, about 4 months old) were set up respectively, and ultrasound physical assisted repair was performed on half of the total number of experimental subjects, and the ultrasound parameters were: ultrasound frequency of 1 MHz, duty cycle of 20%, intensity of 30 mW / cm 2 , once a day for 10 minutes each time, and the remaining experimental subjects did not receive ultrasonic treatment. During the experiment, the management measures of each group were the same. In the fourth and eighth weeks, samples were taken for CT testing to obtain the generated new bone. The results are shown in Figure 6 It can be seen that in the control group, regardless of ultrasound or not, only a thin layer of new bone was generated at the edge of the defect; the material group showed an excellent osteogenesis effect, and new bone was also generated inside the pores as the hydrogel degraded.

[0075] The specific management measures for each group are as follows: Disinfect rabbit cages regularly, at least once a week. Keep cages and cages clean; spray disinfect the indoor space every other day to prevent the spread of bacteria-carrying dust, keep the breeding room quiet and clean, and control the amount of food and water supply. Clean the feed boxes and excrement regularly to avoid moisture and prevent the spread of diseases.

[0076] Example 2

[0077] The same as Example 1, except that in step (1), 0.05 g, 0.1 g and 0.3 g of sodium alginate powder were weighed respectively, and the final concentrations of the sodium alginate solution were 0.005 g / mL, 0.01 g / mL and 0.03 g / mL respectively.

[0078] The hydrogel / calcined bone porous composite scaffold prepared in Example 2 was subjected to an electrical signal test under ultrasound using the method of Application Example 2. The results showed that, under ultrasound, as the concentration of sodium alginate increased, the electrical signal showed a trend of first increasing and then decreasing. This is because the higher the hydrogel monomer concentration, the tighter the internal ion crosslinking, and the limited movement of free ions; the lower the hydrogel monomer concentration, the lower the internal ion crosslinking, and the more free ion movement paths, which is conducive to the differential migration of anions and cations to construct a dynamic electric field.

[0079] Example 3

[0080] The same as Example 1, except that the anhydrous calcium chloride solution in step (2) is replaced by AlCl3 solution, MnCl2 solution, FeCl3 solution, NiCl2 solution and CuCl2·2H2O solution in equal concentrations.

[0081] The hydrogel / calcined bone porous composite scaffold prepared in Example 3 was tested for electrical signals under ultrasound using the method of Application Example 2. The results showed that the electrical signals of the same concentration of materials with different ion sources and sodium alginate after ion crosslinking were not much different, and the performance gap with Example 1 was not much. The only difference was that the hydrogel-calcined bone scaffold crosslinked with calcium chloride was white, while other ion source materials were green, red, etc., which was determined by the properties of the ion source material itself.

[0082] Comparative Example 1

[0083] The same as Example 1, the only difference is that the ultrasonic sound intensity used is different. The ultrasonic frequency in Example 1 is 1 MHz, the duty cycle is 20%, and the intensity is 30 mW / cm 2 Change the frequency to 1MHz, the duty cycle to 20%, and the intensity to 90mW / cm 2 .

[0084] The hydrogel / calcined bone porous composite scaffold prepared in Example 1 was co-cultured with cells using the method of Application Example 4. The results showed that the material was unable to proliferate more cells under ultrasonic response. This may be because there will be a certain temperature increase during the implementation of ultrasound, which has an adverse effect on the cells; excessive sound intensity may also cause cell floating and then cause cell death.

[0085] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a hydrogel / calcined bone porous composite scaffold, characterized in that: The following steps are involved: The calcined bone is combined with a sodium alginate hydrogel material containing free ions by diffusion-mediated method, and then a calcium phosphate mineralization layer is prepared on the surface of the obtained composite material to obtain the hydrogel / calcined bone porous composite scaffold; The preparation of the calcium phosphate mineralized layer specifically comprises soaking the composite material in a dipotassium hydrogen phosphate solution and a calcium chloride solution in sequence; The calcination temperature is 800°C; the calcination time is 4 hours; the free ions are sodium ions and chloride ions; The solution used in the diffusion-mediated method is a calcium chloride solution.

2. The hydrogel / calcined bone porous composite scaffold prepared by the preparation method as claimed in claim 1.

3. The use of the hydrogel / calcined bone porous composite scaffold as claimed in claim 2 as a bone defect repair material, characterized in that: The following steps are involved: The gel / calcined bone porous composite scaffold is used to repair the bone defect, and ultrasound is used as an excitation source to achieve the repair of the bone defect; The frequency of the ultrasonic wave is 1 MHz, the duty cycle is 20%, and the intensity is 10-30 mW / cm 2 .

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