CN111398020B - Method for researching influence of mechanical force on bone - Google Patents
Method for researching influence of mechanical force on bone Download PDFInfo
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- CN111398020B CN111398020B CN201811618809.5A CN201811618809A CN111398020B CN 111398020 B CN111398020 B CN 111398020B CN 201811618809 A CN201811618809 A CN 201811618809A CN 111398020 B CN111398020 B CN 111398020B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N3/00—Investigating strength properties of solid materials by application of mechanical stress
- G01N3/02—Details
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N3/00—Investigating strength properties of solid materials by application of mechanical stress
- G01N3/08—Investigating strength properties of solid materials by application of mechanical stress by applying steady tensile or compressive forces
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N3/00—Investigating strength properties of solid materials by application of mechanical stress
- G01N3/32—Investigating strength properties of solid materials by application of mechanical stress by applying repeated or pulsating forces
- G01N3/34—Investigating strength properties of solid materials by application of mechanical stress by applying repeated or pulsating forces generated by mechanical means, e.g. hammer blows
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Abstract
The invention relates to a method for researching the influence of mechanical force on bones, which consists of a method for resisting loss of mechanical force and a method for promoting bone formation by mechanical force; a method of resisting loss of mechanical force comprising the steps of: ⑴ Preparing an acidic simulated body fluid; ⑵ Preparing an ex-vivo bone sample; ⑶ Fixing an in-vitro bone sample in a mechanical force device, injecting acidic simulated body fluid, applying mechanical force, performing X-ray diffraction technical analysis and a Shelle formula to calculate the average grain size of the apatite crystal; a method of mechanical force promotion of bone formation comprising the steps of: ① Preparing a cell nutrient solution; ② Preparing an osteoblast sample; ③ Fixing osteoblast sample in mechanical force device, injecting cell nutrient solution, applying mechanical force, extracting mRNA and protein, and detecting the expression level of bone formation related factor. The invention is easy to operate, and can control the application frequency and the application time of the mechanical force through a computer, and observe the influence of the mechanical force on the fine structure of the bone sample in vitro in real time.
Description
Technical Field
The invention relates to the technical field of bone research, in particular to a method for researching the influence of mechanical force on bones.
Background
Astronauts experience severe bone loss in space environments, mainly because bone loss rate due to stimulation of lack of earth's attraction of bone tissue under weightlessness condition is greater than bone formation rate. Bone loss can be generated when people lie in bed or lack exercise, and the impact force generated by exercise such as weightlifting, running and the like can improve bone density. These phenomena all indicate that the force has an important role in resisting bone loss and promoting bone formation.
The German medical doctor Wolff has long proposed the law of mechanical strain of bone expressed as: the function of bone is to withstand the mechanical strains of bone tissue during activity and to have the ability to adapt to the needs of these functions. Bone growth is affected by mechanical stimulation to change its structure, and is strong and weak. Fine observation is carried out on main components of bones, namely collagen fibers and apatite crystals by an X-ray crystal diffraction technology, and the fact that the spreading rule of the collagen fibers has an important influence on the local stress and strain relation of compact bones is found, and the crystal grains of the human bone apatite crystals are 5-7 nm larger than 30-80 years old, which indicates that the gradually increased bone loss after the adult period can enable the crystal grains to be smaller. In addition, the osteoblast factors RUNX2 and BMP-2 can participate in signal paths formed by various cytokines, promote osteogenic differentiation and synthesis and secretion of bone matrix, and are closely related to bone formation.
Much research has been carried out on the effect of force on bone. For example, souza (Bone, 2005, 37:810-818) et al in 2005 used mice as the subject, studied the effect of mechanical pressure on living Bone using a small cup with a protective pad, and found that mechanical pressure promoted reconstruction of the tibial cortical Bone of the mice, indicating that proper mechanical pressure could promote Bone formation. However, the research of bones under living conditions has a great limitation that bone changes can be roughly observed only through the bone density measuring instrument and the changes of calcium ion concentration in urine and feces, and the absorption and discharge of calcium are estimated, so that the fine change process of bone tissues and bone cells is difficult to track. Thus, the method of studying the effect of mechanical forces on bone is shifted from in vivo to in vitro. Real-time monitoring of ex-vivo Bone was achieved in the year 2012 by Sun (Bone, 2012, 50:581-591) et al using a mechanical pulling device fixed to an optical microscope, which was found to induce calcium ions to flow out of the Bone matrix and stimulate osteoblast growth, but the device in this study was complex in design and not easy to implement, and did not involve the effect of mechanical forces in resisting Bone loss nor the effects of pressure and impact forces on the fine structure of Bone that are often experienced in human activities. Meanwhile, the conventional two-dimensional planar cell culture technology is difficult to transmit mechanical force to osteoblasts, and is a bottleneck for observing the influence of the mechanical force on the osteoblasts in vitro.
Disclosure of Invention
The invention aims to provide a method for researching the influence of mechanical force on bones, which is simple to operate.
In order to solve the above problems, the method for researching the influence of mechanical force on bones is characterized in that: the method consists of a mechanical force resisting loss method and a mechanical force promoting bone formation method; wherein the method comprises the steps of
The mechanical force resisting loss method comprises the following steps:
⑴ Preparing an acidic simulated body fluid:
Weighing 0.7996 % NaCl,0.0350% NaHCO3,0.0224% KCl,0.0228% K2HPO4･3H2O,0.0305 % MgCl2･6H2O,0.0278% CaCl2,0.0071 % Na2SO4,0.6057% (CH2OH)3CNH2, according to mass percent, sequentially dissolving in distilled water, adopting HCl with the concentration of 1M to adjust the pH value to 6.40, sterilizing by steam at 115 ℃ for 20: 20 min, then placing in a refrigerator refrigerating chamber for preservation, and placing in a water bath at 37 ℃ for 20: 20 min before use;
⑵ Preparing an ex-vivo bone sample:
A small section of polished and flattened fresh bone blocks are cut with a plurality of small openings, and the fresh bone blocks are placed in 0.5M ethylene diamine tetraacetic acid disodium solution for room temperature pretreatment 24 h, so that an isolated bone sample is obtained;
⑶ Fixing the isolated bone sample in a mechanical force device, injecting the acidic simulated body fluid, applying mechanical force, taking the uppermost part of the bone block at the time point to be observed, drying, carrying out ashed treatment, carrying out X-ray crystal diffraction technical analysis, and calculating the average grain size of the apatite crystal by using a Shelle formula;
The mechanical force promoting bone formation method comprises the following steps:
① Preparing a cell nutrient solution:
Preparing an alpha-MEM culture medium containing 20% of fetal bovine serum according to the volume percentage under the aseptic condition, adding penicillin and streptomycin sulfate with the final concentration of 100U/mL and 100 mug/mL respectively, shaking uniformly, placing in a refrigerator refrigerating chamber for preservation, and placing in a water bath at 37 ℃ for 20 min before use;
② Preparation of osteoblast samples:
Soaking a porous apatite-chitosan scaffold with the hydroxyapatite content of 10-20% in 75% alcohol for 15 minutes, and naturally airing in a culture dish under a sterile condition for later use;
According to the volume percentage of 1:9, uniformly mixing osteoblast suspension with the concentration of 2-8 multiplied by 10 7 cells/mL in vitro and 2% low-melting agarose prepared by phosphate buffer solution at 37 ℃, pouring the mixture into a culture dish to submerge a bracket, placing the mixture at 4 ℃ for 20 min, removing redundant low-melting agarose outside the bracket, placing the mixture into the cell nutrient solution, and pre-culturing the mixture at 37 ℃ for 24 h to obtain an osteoblast sample;
③ Fixing the osteoblast sample in a mechanical force device, injecting the cell nutrient solution, applying mechanical force, taking out at the time point required to be observed, blowing and dispersing the osteoblast by using phosphate buffer solution, extracting mRNA and protein, and detecting the expression level of the bone formation related factors.
The mechanical force device in the step ⑶ or the step ③ comprises a stepping motor, a base, a transparent cylindrical container, a bracket, a motor controller and a programmable power supply, wherein the stepping motor, the base, the transparent cylindrical container and the bracket are arranged in an incubator with the temperature of 5% CO 2 and 37 ℃, and the motor controller and the programmable power supply are arranged outside the incubator and sequentially connected with the stepping motor; the stepping motor, the piston and the micrometer are respectively arranged on the bracket; the stepping motor is connected with an elliptical cam through a metal rod, and the bottom of the elliptical cam is provided with the piston; the upper surface and the lower surface of the cylindrical container are both of an opening structure, and the upper part of the side surface of the cylindrical container is provided with a round small hole; a base fixed on the base is arranged in the cylindrical container, and a bone sample is fixed on the base; an elastic gasket is arranged on the bone sample and connected with the piston; the motor controller is connected with a USB interface of a computer, and software matched with the motor controller is installed on the computer.
The bracket is formed by connecting a stainless steel plate, a stainless steel rod and a bolt; the stainless steel plate is provided with a round window, and the round window is provided with the piston which moves up and down.
The piston is provided with a rubber ring.
The elastic gasket is a spring steel sheet with small holes.
The base is provided with a clamping groove for fixing the bone sample.
Compared with the prior art, the invention has the following advantages:
1. The mechanical force device is provided with the micrometer, and the distance between the elliptical cam and the piston can be accurately adjusted through adjusting the bracket according to the micrometer reading, so that the long axis end of the elliptical cam provides uniform and stable mechanical force for a bone sample when contacting the piston each time, and the mechanical force is similar to the pressure and impact force suffered by bones in human activities.
2. The invention can take the isolated bone sample at the required time point to analyze by the X-ray crystal diffraction technique and calculate the average grain size of the apatite crystal by using the Shelle formula, thereby overcoming the defect that the influence of mechanical force on the fine structure of the living bone can not be observed in the prior art.
3. According to the invention, the osteoblast-scaffold complex is prepared by a three-dimensional cell culture technology, so that mechanical force can be successfully transmitted to osteoblasts, and the technical difficulty that the mechanical force is difficult to act on two-dimensional plane cells is solved.
4. The invention can take osteoblast samples at required time points, extract mRNA and protein to detect the expression level of the bone formation related factors, and realize the possibility of in vitro observation of the influence of mechanical force on osteoblasts.
5. The cylindrical container is made of transparent materials, the action process of mechanical force on the bone sample can be directly observed from the outside, and the circular small holes at the upper part of the side surface can be used for exchanging gas and adding liquid inside and outside the container.
6. The elastic gasket is a spring steel sheet with small holes, and the small holes on the elastic gasket can facilitate the flow of liquid.
7. The invention has simple structure and easy operation, can control the application frequency and the application time of mechanical force through a computer, not only provides a reliable research method for clarifying the force capable of promoting bone formation, but also has wide application prospect in the aspect of developing effective bone loss prevention measures.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
Fig. 1 is a front view of a mechanical force device according to the present invention.
Fig. 2 is a side view of the mechanical force device of the present invention.
FIG. 3 is an X-ray crystallography spectrum of an ex-vivo bone sample in an embodiment of the invention.
FIG. 4 shows the mRNA expression levels of the osteoblast bone formation related factors RUNX2 and BMP-2 in the examples of the present invention. # indicates that there was a significant difference (p < 0.05) between the control and mechanical force results.
In the figure: 1-a stepping motor; 2-elliptical cams; 3-a piston; 4-an elastic gasket; 5-micrometer; 6-a cylindrical container; 7, a base; 8, a base; 9-a bracket; 10-bone sample.
Detailed Description
A method for researching the influence of mechanical force on bone is composed of a method for resisting loss of mechanical force and a method for promoting bone formation by mechanical force. Wherein the method comprises the steps of
A method of mechanical resistance to loss comprising the steps of:
⑴ Preparing an acidic simulated body fluid:
0.7996 % NaCl,0.0350% NaHCO3,0.0224% KCl,0.0228% K2HPO4･3H2O,0.0305 % MgCl2･6H2O,0.0278% CaCl2,0.0071 % Na2SO4,0.6057% (CH2OH)3CNH2, by mass percent are weighed and sequentially dissolved in distilled water, the pH value is regulated to 6.40 by adopting HCl with the concentration of 1M, the mixture is sterilized by steam at 115 ℃ for 20 to min and then is placed in a refrigerator refrigerating chamber for storage, and the mixture is placed in a water bath at 37 ℃ for 20 to min before use.
⑵ Preparing an ex-vivo bone sample:
A small section of polished and leveled fresh bone block is scratched with a plurality of small openings and placed in 0.5M Ethylene Diamine Tetraacetic Acid (EDTA) solution for pretreatment at room temperature for 24: 24h, thus obtaining an isolated bone sample.
⑶ The isolated bone sample is fixed in a mechanical force device, acidic simulated body fluid is injected, then mechanical force is applied, the uppermost part of the bone block is taken at the time point required to be observed, and the bone block is dried and ashed to be processed, and then is subjected to X-ray crystal diffraction technical analysis and a Schle formula to calculate the average grain size of the apatite crystal.
A method of mechanical force promotion of bone formation comprising the steps of:
① Preparing a cell nutrient solution:
Preparing an alpha-MEM culture medium containing 20% of fetal bovine serum according to the volume percentage under the aseptic condition, adding penicillin and streptomycin sulfate with the final concentration of 100U/mL and 100 mug/mL respectively, shaking uniformly, placing in a refrigerator refrigerating chamber for preservation, and placing in a water bath at 37 ℃ for 20 min before use;
② Preparation of osteoblast samples:
Soaking a porous apatite-chitosan scaffold with the hydroxyapatite content of 10-20% in 75% alcohol for 15 minutes, and naturally airing in a culture dish under a sterile condition for later use;
According to the volume percentage of 1:9, uniformly mixing osteoblast suspension with the concentration of 2-8 multiplied by 10 7 cells/mL in vitro and 2% low-melting agarose prepared by phosphate buffer solution at 37 ℃, pouring into a culture dish to submerge a bracket, placing at 4 ℃ for 20 min, removing redundant low-melting agarose outside the bracket, placing into cell nutrient solution, and pre-culturing at 37 ℃ for 24 h to obtain an osteoblast sample;
③ Fixing osteoblast sample in mechanical force device, injecting cell nutrient solution, applying mechanical force, taking out at the time point to be observed, blowing and dispersing osteoblast with phosphate buffer solution, extracting mRNA and protein, and detecting the expression level of bone formation related factors.
The mechanical force device comprises a stepping motor 1, a base 8, a transparent cylindrical container 6, a bracket 9 which are arranged in an incubator with 5% CO 2 and 37 ℃, and a motor controller and a programmable power supply which are arranged outside the incubator and are sequentially connected with the stepping motor 1.
The bracket 9 is respectively provided with a stepping motor 1, a piston 3 and a micrometer 5; the bracket 9 is formed by connecting a stainless steel plate, a stainless steel rod and a bolt; the stainless steel plate is provided with a circular window provided with a piston 3 moving up and down. The piston 3 is made of aluminum alloy material, and is provided with a rubber ring. The stepping motor 1 is connected with an elliptical cam 2 through a metal rod, and the bottom of the elliptical cam 2 is provided with a piston 3; the upper and lower surfaces of the cylindrical container 6 are both of an opening structure, and the upper part of the side surface of the cylindrical container is provided with a round small hole; a base 7 fixed on a base 8 is arranged in the cylindrical container 6, and a bone sample 10 is fixed on the base 7; the base 7 is provided with a clamping groove for fixing the bone sample 10. The bone sample 10 is provided with an elastic gasket 4, and the elastic gasket 4 is connected with the piston 3; the elastic pad 4 is a spring steel sheet with small holes (as shown in fig. 1-2). The motor controller is connected with a USB interface of a computer, and the computer is provided with software matched with the motor controller.
The working principle of the mechanical force device in the invention is as follows: the stepping motor 1 drives the elliptical cam 2 to periodically act on the piston 3, and the micrometer 5 can adjust the distance between the elliptical cam 2 and the piston 3, so that stronger mechanical force is generated when the long shaft end of the elliptical cam 2 presses the piston 3, and no mechanical force is generated when the short shaft end contacts the piston 3; the piston 3 transmits the mechanical forces to which it is subjected to the bone sample 10 through the resilient pad 4, similar to the pressure and impact forces to which the bone is subjected during human activity.
Example 1 takes as an example the effect of in vitro observation of mechanical forces on the fine structure of an isolated bone apatite crystal, the following is given in particular embodiments:
[ preparation of an acid-simulated body fluid ]
0.7996 % NaCl,0.0350% NaHCO3,0.0224% KCl,0.0228% K2HPO4･3H2O,0.0305 % MgCl2･6H2O,0.0278% CaCl2,0.0071 % Na2SO4,0.6057% (CH2OH)3CNH2, By mass percent are weighed and sequentially dissolved in distilled water, the pH value is regulated to 6.40 by adopting HCl with the concentration of 1M, the mixture is sterilized by steam at 115 ℃ for 20 to min and then is placed in a refrigerator refrigerating chamber for storage, and the mixture is placed in a water bath at 37 ℃ for 20 to min before use.
The acidic simulated body fluid can maintain the bone loss state of the isolated bone.
[ Preparation of bone sample ]
A small section of fresh pig bone which is purchased in the market is stripped of surface connective tissue, polished and leveled to prepare two bone blocks with the same texture, size and shape (length multiplied by width multiplied by height is about 5cm multiplied by 1 cm). Several small openings were made in the bone pieces to increase the contact area with the liquid, and the pieces were pre-treated 24. 24 h in 0.5M disodium ethylenediamine tetraacetate (EDTA) solution at room temperature to create a slight bone loss state and increase the elasticity of the bone pieces when subjected to mechanical forces. One was used to observe the effect of mechanical force on the fine structure of the isolated bone apatite crystals, and one was placed under the same conditions but no impact force was applied as a control.
[ Operation procedure ]
⑴ The prepared bone sample 10 is fixed in the clamping groove of the base 7 so as to keep the vertical state.
⑵ The cylindrical container 6 is placed on the base 8 with the bone sample 10 placed therein. After fixing the bottom of the sealed cylindrical container 6 with transparent glass cement, the elastic pad 4 and the piston 3 are placed on the bone sample 10 in sequence, and the acidic simulated body fluid is slowly injected until the bone sample 10 is immersed.
⑶ The bracket 9 is mounted so that the piston 3 is placed in a circular window of stainless steel plate. The stepping motor 1 and the oval cam 2 connected with the stepping motor are arranged on the bracket, and the distance between the oval cam 2 and the piston 3 is adjusted through the micrometer 5, so that the long axis end of the oval cam 2 provides uniform and stable mechanical force for the bone sample 10 when contacting the piston 3 each time.
⑷ And (3) turning on a power supply, starting a computer, adjusting the application frequency and the application time of the mechanical force, and starting timing.
[ Bone sample detection ]
The uppermost portion of the bone pieces was dried and ashed after application of mechanical force 60 h, and then analyzed by scanning with an X-ray crystallography (model DRON-4-07, burevestnik). Phase analysis using international standard JCPDS (Joint Committee on Powder Diffraction Standards), data analysis was performed using DifWin-1 (metalon-TC) software package, and the resulting X-ray crystal diffraction pattern of bone sample 10 is shown in fig. 3. Meanwhile, calculating the average grain size (L) of the apatite crystal by using a Schle formula:
Wherein: lambda is the wavelength of the X-ray, ϑ is the diffraction angle, beta m is the linewidth of the pure diffraction spectrum obtained from small grains, and K is the crystal shape dependent constant. The grain parameter of the bone sample 10 to which the mechanical force was applied was 35 nm, and the grain parameter of the bone sample 10 to which the mechanical force was not applied was 27 nm. The mechanical force action is shown to maintain the structure and size of bone tissue apatite crystals so as to effectively resist bone loss caused by environmental changes.
Example 2 is an example of the effect of in vitro mechanical force on the change in the expression of osteoblast bone formation related factors, and is specifically described as follows:
[ preparation of cell nutrient solution ]
Preparing an alpha-MEM culture medium containing 20% of fetal bovine serum according to the volume percentage under the aseptic condition, adding penicillin and streptomycin sulfate with final concentrations of 100U/mL and 100 mug/mL respectively, shaking uniformly, placing in a refrigerator refrigerating chamber for storage, and placing in a water bath at 37 ℃ for 20 min before use.
The cell nutrient solution can maintain the cell activity of osteoblasts in the hydroxyapatite-chitosan scaffold.
[ Preparation of osteoblast samples ]
Two porous apatite-chitosan scaffolds with the hydroxyapatite content of 10-20% and the length, width and height of 5cm multiplied by 1cm are soaked in 75% alcohol for 15 minutes, placed in a culture dish with the diameter of 6 cm, and naturally dried under aseptic conditions for standby.
10 Bottles of well-grown mouse MC3T3-E1 osteoblasts (purchased from cell banks of the national academy of sciences) were taken, washed with phosphate buffer, added with 0.05% pancreatin (containing EDTA-2 Na), digested at 37 ℃ for 5 min, and prepared into a suspension with a concentration of 8X 10 7 cells/mL with cell nutrient solution.
1 ML osteoblast suspension and 2% low-melting agarose (HyAgaroseTM) prepared from 9 mL phosphate buffer solution are taken and mixed uniformly at 37 ℃, poured into a culture dish to submerge a bracket, and redundant bubbles are sucked by a suction tube.
Placing at 4deg.C for 20min, removing redundant low-melting agarose, placing into cell nutrient solution, and pre-culturing at 37deg.C for 24 h to obtain osteoblast sample.
Taking an osteoblast sample, placing the osteoblast sample in a mechanical force device, injecting a cell nutrient solution, and applying mechanical force; the other block was placed under the same conditions but no mechanical force was applied as a control.
[ Osteoblast sample detection ]
Taking out osteoblast samples after applying mechanical force 24h, blowing MC3T3-E1 osteoblast in the bracket by using phosphate buffer solution, dispersing, adding Trizol lysate, placing on ice for 5-min, and preserving at-80 ℃. After the sample to be detected is collected, RT-PCR is used for detecting the expression of bone formation related factors RUNX2 and BMP-2, GAPDH is used as an internal reference for calibration, and a 2-fating Ct method is used for analyzing the gene expression.
FIG. 4 shows the mRNA expression levels of the bone formation related factors RUNX2 and BMP-2 of MC3T3-E1 osteoblasts. Under the condition of applying mechanical force, the mRNA levels of RUNX2 and BMP-2 are respectively increased by 17.35 percent and 23.93 percent, and compared with a control without applying mechanical force, the mRNA levels have obvious difference (p is less than 0.05), which shows that the mechanical force can improve the expression of the bone formation related factors in osteoblasts and has the effect of promoting the bone formation.
Claims (5)
1. A method for studying the effect of mechanical force on bone, characterized by: the method consists of a mechanical force resisting loss method and a mechanical force promoting bone formation method; wherein the method comprises the steps of
The mechanical force resisting loss method comprises the following steps:
⑴ Preparing an acidic simulated body fluid:
Weighing 0.7996 % NaCl,0.0350% NaHCO3,0.0224% KCl,0.0228% K2HPO4･3H2O,0.0305 % MgCl2･6H2O,0.0278% CaCl2,0.0071 % Na2SO4,0.6057% (CH2OH)3CNH2, according to mass percent, sequentially dissolving in distilled water, adopting HCl with the concentration of 1M to adjust the pH value to 6.40, sterilizing by steam at 115 ℃ for 20: 20 min, then placing in a refrigerator refrigerating chamber for preservation, and placing in a water bath at 37 ℃ for 20: 20 min before use;
⑵ Preparing an ex-vivo bone sample:
A small section of polished and flattened fresh bone blocks are cut with a plurality of small openings, and the fresh bone blocks are placed in 0.5M ethylene diamine tetraacetic acid disodium solution for room temperature pretreatment 24 h, so that an isolated bone sample is obtained;
⑶ Fixing the isolated bone sample in a mechanical force device, injecting the acidic simulated body fluid, applying mechanical force, taking the uppermost part of the bone block at the time point to be observed, drying, carrying out ashed treatment, carrying out X-ray crystal diffraction technical analysis, and calculating the average grain size of the apatite crystal by using a Shelle formula; the mechanical force device comprises a stepping motor (1) arranged in an incubator with the volume concentration of 5% CO 2 and the temperature of 37 ℃, a base (8), a transparent cylindrical container (6), a bracket (9) and a motor controller and a programmable power supply which are arranged outside the incubator and sequentially connected with the stepping motor (1); the stepping motor (1), the piston (3) and the micrometer (5) are respectively arranged on the bracket (9); the stepping motor (1) is connected with an elliptical cam (2) through a metal rod, and the bottom of the elliptical cam (2) is provided with the piston (3); the upper surface and the lower surface of the cylindrical container (6) are both of an opening structure, and the upper part of the side surface of the cylindrical container is provided with a round small hole; a base (7) fixed on the base (8) is arranged in the cylindrical container (6), and a bone sample (10) is fixed on the base (7); an elastic gasket (4) is arranged on the bone sample (10), and the elastic gasket (4) is connected with the piston (3); the motor controller is connected with a USB interface of a computer, and the computer is provided with software matched with the motor controller;
The mechanical force promoting bone formation method comprises the following steps:
① Preparing a cell nutrient solution:
Preparing an alpha-MEM culture medium containing 20% of fetal bovine serum according to the volume percentage under the aseptic condition, adding penicillin and streptomycin sulfate with the final concentration of 100U/mL and 100 mug/mL respectively, shaking uniformly, placing in a refrigerator refrigerating chamber for preservation, and placing in a water bath at 37 ℃ for 20 min before use;
② Preparation of osteoblast samples:
Soaking a porous apatite-chitosan scaffold with the hydroxyapatite content of 10-20% in 75% alcohol for 15 minutes, and naturally airing in a culture dish under a sterile condition for later use;
According to the volume percentage of 1:9, uniformly mixing osteoblast suspension with the concentration of 2-8 multiplied by 10 7 cells/mL in vitro and 2% low-melting agarose prepared by phosphate buffer solution at 37 ℃, pouring the mixture into a culture dish to submerge a bracket, placing the mixture at 4 ℃ for 20 min, removing redundant low-melting agarose outside the bracket, placing the mixture into the cell nutrient solution, and pre-culturing the mixture at 37 ℃ for 24 h to obtain an osteoblast sample;
③ Fixing the osteoblast sample in a mechanical force device, injecting the cell nutrient solution, applying mechanical force, taking out at the time point required to be observed, blowing and dispersing the osteoblast by using phosphate buffer solution, extracting mRNA and protein, and detecting the expression level of the bone formation related factors.
2. A method of studying the effect of mechanical force on bone as claimed in claim 1, wherein: the bracket (9) is formed by connecting a stainless steel plate, a stainless steel rod and a bolt; the stainless steel plate is provided with a circular window provided with the piston (3) which moves up and down.
3. A method of studying the effect of mechanical force on bone as claimed in claim 1, wherein: the piston (3) is provided with a rubber ring.
4. A method of studying the effect of mechanical force on bone as claimed in claim 1, wherein: the elastic gasket (4) is a spring steel sheet with small holes.
5. A method of studying the effect of mechanical force on bone as claimed in claim 1, wherein: the base (7) is provided with a clamping groove for fixing the bone sample (10).
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