Physical method for regulating and controlling activity of osteoclast

Abstract

The invention provides a physical method for regulating and controlling the activity of osteoclasts. The physical method is used for regulating and controlling the activity of the osteoclasts. If the target is to inhibit the differentiation of the osteoclast, pulse electricity with first electric field intensity is output to the mononuclear macrophage, so that the differentiation of the mononuclear macrophage to the osteoclast is reduced; and if the target is to enhance osteoclast differentiation, outputting pulse electricity with second electric field intensity to the mononuclear macrophages, so that osteoclast differentiation of the mononuclear macrophages is enhanced. Wherein the intensity of the first electric field is not greater than 50V / m, and the intensity of the second electric field is 75-150V / m. The method provided by the invention is a physical method capable of directly regulating and controlling the osteoclast differentiation process, and the aim of regulating and controlling the osteoclast activity can be achieved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a physical method for regulating osteoclast activity. Background Art

[0002] As the main component of the skeletal system, bone tissue completes bone remodeling through continuous division and apoptosis to maintain the relative homeostasis of the skeletal system. Osteoclasts, as an important type of cell in bone tissue, remove aged or damaged bone tissue by dissolving and absorbing bone.

[0003] Osteoclasts are differentiated from mononuclear macrophages under the action of cytokines. Since the differentiation cycle of osteoclasts is short and mature osteoclasts are terminal cells that rapidly apoptosis after exercising their functions, they are often regulated by drugs. For example, bisphosphonates are used to inhibit osteoclast activity. It can be seen that the prior art lacks a physical method that can directly regulate its differentiation process. Summary of the invention

[0004] The present application provides a physical method and device for regulating osteoclast differentiation, which are used to physically regulate the process of target mononuclear macrophages differentiating into osteoclasts, so as to achieve the purpose of regulating osteoclast activity.

[0005] The present application provides a control method, comprising:

[0006] Receiving a control instruction; wherein the control instruction refers to a control type;

[0007] Determine whether the regulation type is to inhibit osteoclast activity; if so, output pulse electricity with a first electric field strength to the mononuclear macrophages to reduce the osteoclast differentiation of the cells; wherein the first electric field strength is not greater than 50V / m.

[0008] The control device provided in the embodiment of the present application determines the electric field strength of the output pulse electricity by judging the control type in the received control instruction. The purpose of regulating the activity of osteoclasts is achieved by the electric field strength corresponding to the control type. When the target is determined to be to inhibit the activity of osteoclasts, an electric stimulation with an electric field strength of no more than 50V / m is directly output to inhibit the differentiation of target cells into osteoclasts, thereby achieving the purpose of inhibiting the activity of osteoclasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 A schematic diagram of the structure of an osteoclast differentiation and culture device provided in an embodiment of the present application;

[0010] Figure 2 A statistical graph showing the ratio of live cells to dead cells at different graphite powder concentrations provided in an embodiment of the present application;

[0011] Figure 3 A schematic diagram of pulse electricity output by a control device provided in an embodiment of the present application;

[0012] Figure 4 This is a CCK8 activity test diagram of cells under pulse electrical stimulation provided in an embodiment of the present application;

[0013] Figure 5 A statistical graph of osteoclast area after TRAP staining provided in an embodiment of the present application;

[0014] Figure 6 The optical microscopic images and electron microscopic images of osteoclasts after pulsed electrical stimulation under different electric field intensities provided in the embodiments of the present application; wherein, (a)-(e) are optical microscopic images of osteoclasts on a cell culture slide in a cell culture device after TRAP staining; (A)-(E) are scanning electron microscopic images (SEM) of osteoclasts on a bovine bone slice after pulsed electrical stimulation;

[0015] Figure 7 A statistical graph of test results of qPCR verification of osteoclast membrane proteins OSCAR and RANK provided in an embodiment of the present application;

[0016] Figure 8 A statistical graph of test results of qPCR verification of osteoclast NFATc1 provided in an embodiment of the present application;

[0017] Fig. 9 A statistical graph of test results of qPCR verification of osteoclast exocrine factor provided in an embodiment of the present application;

[0018] Fig.10 A statistical graph of test results of qPCR verification of osteoclast fusion protein DC-STAMP provided in an embodiment of the present application;

[0019] Fig.11 This is a diagram of cell growth density near the anode and cathode regions on the cell culture slide when the control device provided in an embodiment of the present application outputs pulse electricity to osteoclasts on the cell culture slide in the cell culture device. DETAILED DESCRIPTION

[0020] In view of the problem that there is a lack of physical methods to regulate the osteoclast differentiation process in the prior art, the embodiment of the present application provides a physical method for regulating osteoclast differentiation, which is based on the instruction to regulate the up-regulation or down-regulation of osteoclast activity, and outputs an electric field of corresponding strength to mononuclear macrophages. When the regulation type is determined to inhibit activity, pulse electricity with an electric field strength of no more than 50V / m is output to the target cells to inhibit the differentiation of mononuclear macrophages into osteoclasts, so as to achieve the purpose of inhibiting osteoclast activity. When the regulation type is to promote osteoclast activity, an electric signal with an electric field strength between 75-150V / m is input to the mononuclear macrophages to promote the differentiation of mononuclear macrophages into osteoclasts, so as to achieve the purpose of promoting osteoclast activity.

[0021] In order to better understand the above technical scheme, the technical scheme of the present application is described in detail below through the accompanying drawings and specific embodiments. It should be understood that the embodiments of the present application and the specific features in the embodiments are detailed descriptions of the technical scheme of the present application, rather than limitations on the technical scheme of the present application. In the absence of conflict, the embodiments of the present application and the technical features in the embodiments can be combined with each other.

[0022] The embodiments of the present application provide a physical method for regulating the activity of osteoclasts. Given a regulation target, the cell activity of the monocyte macrophages to be regulated is compared with the regulation target to determine the regulation direction. If the regulation direction is to inhibit the differentiation of osteoclasts, pulse electricity with a first electric field strength is output to the monocyte macrophages to reduce the differentiation of the monocyte macrophages into osteoclasts. If the regulation direction is to enhance the differentiation of osteoclasts, pulse electricity with a second electric field strength is output to the monocyte macrophages to enhance the differentiation of the monocyte macrophages into osteoclasts. The first electric field strength is not greater than 50 V / m, and the second electric field strength is between 75 and 150 V / m.

[0023] In some embodiments, the first electric field strength is 20-50 V / m.

[0024] In some embodiments, the second electric field strength is between 75-150 V / m; preferably 75-125 V / m.

[0025] In some embodiments, the pulse electricity is a direct current with a duty cycle of not less than 50% and a frequency of not less than 1 Hz. The frequency of the pulse electricity may be 1 Hz-20 kHz.

[0026] In some embodiments, the method for regulating osteoclast activity is to culture the cells in a cell-friendly environment, i.e., a cell culture incubator at 37° C. in a humidified environment of 5% CO 2 .

[0027] See also Figure 1, a schematic diagram of the structure of an osteoclast differentiation culture device is provided; the device is composed of a graphite electrode 1, a cell culture well plate cover 2, a cell culture well plate 3, a cell culture climbing sheet 4, and a wire 5. Among them, the (upper) surface of the cell culture climbing sheet 4 facing the cell culture well plate cover 2 is used to culture cells 6.

[0028] Specifically, the graphite electrode 1 passes through the cell culture well plate cover 2 and is connected to the bottom of the cell culture well plate 3, and the other end is connected to the wire 5, and the wire 5 is connected to the signal generator. A cell culture sheet 4 adapted to the distance between the graphite electrode 1 is placed at the bottom of the cell culture well plate 3, and cells 6 are planted on the cell culture sheet 4. Among them, the size of the graphite electrode 1 is 1mm*5mm*30mm. The cell culture well plate 3 is a standard 24-well plate. The cell culture sheet 4 is a square with a side length of 5mm. The wire 5 is fixedly connected to one end of the graphite electrode 1 by copper tape. The graphite electrode 1 is fixed to the cell culture well plate cover 2 by hot melt adhesive.

[0029] The distance between the two graphite electrodes 1 is d, and the pulse DC voltage output by the signal generator is U. Then the electric field strength felt by the cells between the two electrodes is E=U / d.

[0030] For ease of understanding, the following are the steps for osteoclasts to perform bone phagocytosis:

[0031] The bone phagocytosis function of osteoclasts mainly includes three steps: differentiation of monocytes and macrophages into osteoclast precursor cells, fusion of osteoclast precursors, and release of enzymes by osteoclasts to corrode bone.

[0032] Among them, the protein pathways of precursor cell differentiation include: RANKL (Receptor activator of nuclear factor kappa-Βligand) / RANK (Receptor activator of nuclear factor kappa-Β) / MAPKs (mitogen-activated protein kinase) / NF-κB (nuclear factor kappa-B) / NFATc1 (Nuclear Factor Of Activated TCells 1), RANK / PLCγ (Phospholipase Cγ, Programmable Logic Controllerγ) / Ca 2+ / NFATc1 and OSCAR (osteoclast-associated receptor) / SYK (Spleen Tyrosine Kinase) / PLCγ / Ca 2+ / NFATc1.

[0033] The protein pathways required for osteoclast precursor fusion include: DC-STAMP (Dendritic Cell-Specific Transmembrane Protein), uPAR (migration-associated protein urokinase-type plasminogen activator receptor) / phosphatidylinositol kinase-serine / PI3k-Akt (Phosphatidylinositide 3-kinases-protein kinase B), BMP-2 (bone morphogenetic protein 2), CD47 (cluster of differentiation 47) and Syncytin1 required for osteoclast precursor recognition, and Pyk2 (Proline-rich tyrosine kinase 2) / c-Src (Proto-oncogene tyrosine-protein kinase) required for osteoclast function. Src) / c-CbI (E3 ubiquitin ligase, casitas B-lineage lymphoma), integrin ανβ3 and released CTSK (cathepsin K), H+, MMP-9 (matrix metalloproteinase-9), RANKL (receptor activator of nuclear factor kappa-Βligand).

[0034] Furthermore, since the effect of graphite on cells mainly comes from the graphite powder particles that fall off during the immersion of graphite in the cell culture medium, different concentrations of graphite were added to the cell culture medium to form a concentration gradient of 0-500 μg / mL, and fibroblasts (L929 cells, which are used for toxicity testing) were cultured. When the cells grew for 24 hours, the cells were stained for live and dead cells, and the statistical results of the number of live cells and the number of dead cells were obtained as shown in the figure. Figure 2 As shown. Figure 2 It can be seen that graphite electrodes will not affect the activity of cells and have good cell compatibility. Other conductive materials with good biocompatibility can also be used as electrodes, such as precious metals such as Pt and Au.

[0035] Further, the primary mononuclear macrophages were stimulated by outputting a DC pulse with increasing gradient, and the voltage not exceeding 20V (e.g., 1.5V) was stimulated for 60 minutes every day at 1Hz, a duty cycle of 50%, and a voltage not exceeding 20V. The cell viability test of CCK8 was performed on the cells after the electrical stimulation on the 1st, 3rd, and 5th days, respectively. Figure 3 The test results are as follows: Figure 4 As shown. Figure 4 It can be seen that as the voltage intensity increases, the electrical stimulation intensity of osteoclasts increases, and the activity of osteoclasts shows a slightly downward trend. Moreover, under the stimulation of 150V / m electric field intensity, the cell viability can still be maintained at more than 80%. Therefore, within the voltage intensity range of 0-150V / m, pulse electrical stimulation has almost no effect on cell activity.

[0036] The above primary mononuclear macrophages are derived from: first, use scissors and forceps sterilized at high temperature and high pressure to collect 1xPBS from the tibia and femur of the hind limbs of mice killed by dislocation of the neck and blow out the bone marrow. Then centrifuge the suspension at 1000rpm for 5min and remove the supernatant. Use a pipette to absorb 3ml of red blood cell lysis solution and blow the cells apart. After standing at room temperature for 3min, centrifuge at 1000rpm for 3min and remove the supernatant. Then, use 1ml of α-MEM complete medium containing 10% FBS and 1% double antibody to blow the cells apart, and culture the cells at 37°C with a complete culture base of this component ratio. Collect the supernatant after 12h, configure the supernatant to contain 50ng / ml M-CSF concentration and culture for 72h, and collect the adherent cells with a cell scraper as primary mononuclear macrophages.

[0037] Osteoclast differentiation culture: Use the aforementioned osteoclast culture device to culture primary mononuclear macrophages. When the primary mononuclear macrophages grow to a density of 80%-90%, collect the cells with a scraper and resuspend them into single cells. 5 / well density was spread on the climbing sheet in the medium-well plate of the aforementioned osteoclast culture device, and cultured at 37°C with α-MEM complete culture medium containing 50ng / ml M-CSF and 100ng / ml RANKL, and the medium was changed every two days. Osteoclasts are multinuclear cells formed by the fusion of multiple mononuclear macrophages. Compared with precursor cells, osteoclasts have larger actin rings (that is, cell area). The larger the cell area, the larger the absorption pit formed on the bone surface, and the cell area is positively correlated with the bone absorption capacity of osteoclasts.

[0038] Therefore, in this embodiment, the area of ​​osteoclasts is used to determine the differentiation of osteoclasts in the target bone tissue after the corresponding pulse electricity is output to the target bone tissue. Specifically, in the process of inducing osteoclast differentiation, pulse electricity is applied for 2 hours every 24 hours, and repeated multiple times, such as for 3 days; or, pulse electricity is applied for 1 hour every 12 hours, and continued for multiple days, such as repeated 6 times; then, on the 5th day of inducing osteoclast differentiation, the cells are stained according to the instructions of the tartrate-resistant acid phosphatase TRAP kit, and the staining results are shown in Figure 6 (a)-(e). In TRAP staining, large cells with obvious actin rings are osteoclasts. The area of ​​osteoclasts is counted and plotted. Figure 6 . Figure 6 (a)-(e) are light microscopic images of osteoclasts under pulsed electrical stimulation with different electric field intensities, while other parameters are the same (duty cycle is 50%, frequency is 1Hz). The electric field intensities corresponding to (a)-(e) are 0V / m, 20V / m, 50V / m, 75V / m, and 150V / m, respectively. Figure 6 From (a) to (e), it can be seen that when the electric field strength is less than 75 V / m, the pulsed electrical stimulation has an inhibitory effect on the activity of osteoclasts; and when the electric field strength is greater than or equal to 75 V / m, the pulsed electrical stimulation has a promoting effect on the activity of osteoclasts. Figure 6 In (a)-(e), Figure 5 It can be seen that within the electric field strength range of 20-50V / m, the differentiation of osteoclasts is significantly inhibited; when the electric field strength is 75-150V / m, especially when it is equal to 75V / m, the differentiation of osteoclasts is significantly promoted.

[0039] Furthermore, RNA was extracted from osteoclasts on the 5th day of culture using an RNA extraction kit, and then qPCR was performed to verify the osteoclast membrane proteins OSCAR and RANK based on the designed primers. The test results are shown in Figure 7 ; qPCR validation of osteoclast NFATc1, test results see Figure 8 ; qPCR validation of osteoclast exocrine factors, test results see Fig. 9 ; qPCR verification of cell fusion protein DC-STAMP, test results see Fig.10 .

[0040] Depend on Figure 7-Figure 10 Can be obtained, compared to Figure 7 , Fig.10 The mRNA expression levels of the experimental group and the 0V / m group were significantly different. DC-STAMP protein, as a fusion protein, led osteoclast precursors to fuse and differentiate into mature osteoclasts. Fig. 9The secretion factors of mature osteoclasts showed that DC-STAMP was correlated with the secretion factors.

[0041] Continue to refer Figure 7-Figure 10 The effect of 0-150V / m electrical stimulation on the osteoclast membrane proteins OSCAR and RANK was not obvious, while the nuclear transcription-related gene NFATc1 showed a strong dependence on the current intensity. The nuclear secretion factor and cell fusion protein DC-STAMP also showed no obvious response to current stimulation, and the expression levels of the nuclear secretion factor and cell fusion protein DC-STAMP showed the same trend under electrical stimulation.

[0042] Fig.11 The optical microscope image of the osteoclast growth density in the area near the graphite electrode (yin and yang electrodes) on the cell culture slide when the wire 5 and graphite electrode 1 of the cell culture device output electrical stimulation to the osteoclasts. The area near the graphite electrode is the area on both sides of the cell culture slide connected to the graphite electrode. Fig.11 It can be obtained that under the electrical stimulation of 0-150V / m, osteoclasts show a tendency to migrate toward the negative electrode.

[0043] So we can get:

[0044] (1) Since the DC-STAMP protein acts as a fusion protein to direct the fusion and differentiation of osteoclast precursors into mature osteoclasts, the method for regulating osteoclast activity provided in the embodiments of the present application affects cell migration and DC-STAMP-mediated cell-to-cell fusion by outputting pulse electricity, thereby affecting the differentiation of mature osteoclasts, and thus affecting the secretion of factors by osteoclasts, thereby affecting the osteoclasts' bone phagocytic function.

[0045] (2) Electrical stimulation regulates intracellular Ca through the response of intracellular PLCγ 2+ concentration, thereby affecting NFATc1 / Ca 2+ The main axis of osteoclast differentiation is mediated by calmodulin, which regulates the self-amplification of nuclear NFATc1, making NFATc1 show current intensity dependence.

[0046] (3) After determining the regulation type and outputting the corresponding pulse electricity, the method provided in the embodiment of the present application stimulates Rac1 / NOx / NF-κB to make it migrate in response to the electrical signal and activate the intracellular reactive oxygen species to affect the intracellular Ca 2+ Mobilization of storage can achieve the purpose of regulating osteoclast differentiation.

[0047] The above Cck8 activity test, TRAP staining, RNA extraction and transcription fluorescence quantitative test were performed according to the reagent instructions.

[0048] Further, a bone model was constructed by making a cow bone slice with a structure similar to that of human bones. The cow bone slice was subjected to DC pulse electrical stimulation with different electric field strengths; the frequency was 1 Hz, and the duty cycle was 50%. Then, a scanning electron microscope (SEM) test was performed on the mononuclear macrophages implanted on the cow bone slice that received the aforementioned electrical stimulation, and the SEM images were obtained as shown in the following figure. Figure 6 As shown in AE. The electric field strengths corresponding to (A)-(E) are 0V / m, 20V / m, 50V / m, 75V / m, and 150V / m, respectively. Figure 6 As shown in Figures AE, pits of varying degrees appeared on the surface of the bovine bone slices. In (A), under the stimulation of an electric field strength of 0V / m, although pits with larger areas appeared, the depths of all pits were lower than the depths of the pits in (B)-(E). In (D), under the stimulation of an electric field strength of 75V / m, the pits on the bovine bone slices had the characteristics of large area and deep depth. It can be seen that under the condition of an electric field strength of 75V / m, osteoclasts will form closed absorption rings after differentiation and maturation on the surface of the bone slice, and then secrete acidic substances to degrade the bone, and finally form absorption pits on the surface of the bone slice.

[0049] See Table 1 for the sources of the above test reagents, and see Table 2 for the primer sequences involved in the PCR test.

[0050] Table 1

[0051]

[0052] Among them, HiScript III RT SuerMix for qPCR, Taq Pro Universal SYBR qPCRMix, and ddH2O are reagents used in PCR tests.

[0053] Table 2 Primer sequences

[0054]

[0055] Based on the same inventive concept, the present application embodiment provides a device for regulating osteoclast activity. The device corresponds to the method for regulating osteoclasts. The specific implementation of the device can be found in the description of the aforementioned method embodiment, and the repeated parts will not be repeated. The device includes:

[0056] Cell culture unit: used to culture mononuclear macrophages to be regulated;

[0057] Receiving and comparing unit: used to receive the regulation target; compare the cell activity of the mononuclear macrophage to be regulated with the regulation target to determine the regulation direction;

[0058] Pulse applying unit: used to apply pulse electricity to the mononuclear macrophages to be regulated. If the regulation direction is to inhibit the differentiation of osteoclasts, pulse electricity with a first electric field strength is output to the mononuclear macrophages to reduce the differentiation of the mononuclear macrophages into osteoclasts; if the regulation direction is to enhance the differentiation of osteoclasts, pulse electricity with a second electric field strength is output to the mononuclear macrophages to enhance the differentiation of the mononuclear macrophages into osteoclasts. The duty cycle of the pulse electricity provided by the pulse applying unit is not less than 50%, and the pulse electricity is direct current with a frequency of not less than 1 Hz.

[0059] The pulse power provided by the pulse applying unit is direct current with a frequency range of 1 Hz-20 kHz and a duty cycle of not less than 50%.

[0060] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A physical method for regulating osteoclast activity, characterized in that: Given a control target, if the control target is to inhibit the differentiation of osteoclasts, outputting pulse electricity with a first electric field strength to the mononuclear macrophages to reduce the differentiation of the mononuclear macrophages into osteoclasts; If the control target is to enhance osteoclast differentiation, pulse electricity with a second electric field strength is output to the mononuclear macrophages to enhance the osteoclast differentiation of the mononuclear macrophages, wherein the first electric field strength is not greater than 50V / m and the second electric field strength is between 75-150V / m.

2. The method according to claim 1, characterized in that The first electric field strength ranges from 20 to 50 V / m.

3. The method according to claim 1, characterized in that The second electric field strength ranges from 75 to 125 V / m.

4. The method according to any one of claims 1 to 3, characterized in that: The frequency range of the pulse electricity is 1 Hz-20 kHz.

5. The method according to any one of claims 1 to 4, characterized in that: The pulse electricity has a duty cycle of no less than 50% direct current.

6. The method according to any one of claims 1 to 5, characterized in that: The pulse electricity is applied for 2 hours every 24 hours, and repeated multiple times; or, the pulse electricity is applied for 1 hour every 12 hours, and repeated multiple times.

7. The method according to any one of claims 1 to 6, characterized in that: In 5% CO 2 The cells were cultured in a gas-humidified cell culture incubator.

8. The method according to any one of claims 1 to 7, characterized in that: Monocytes and macrophages were cultured in α-MEM complete medium containing 50 ng / ml M-CSF and 100 ng / ml RANKL in a cell culture incubator at 37°C.

9. A device for regulating osteoclast activity, characterized in that: The device includes: A cell culture unit, used for culturing mononuclear macrophages to be regulated; A receiving and comparing unit, used for receiving a regulation target; comparing the cell activity of the mononuclear macrophage to be regulated with the regulation target to determine the regulation direction; A pulse applying unit is used to apply pulse electricity to the mononuclear macrophages to be regulated; if the regulation direction is to inhibit the differentiation of osteoclasts, pulse electricity with a first electric field strength is output to the mononuclear macrophages to reduce the differentiation of the mononuclear macrophages into osteoclasts; if the regulation direction is to enhance the differentiation of osteoclasts, pulse electricity with a second electric field strength is output to the mononuclear macrophages to enhance the differentiation of the mononuclear macrophages into osteoclasts; wherein the first electric field strength is not greater than 50V / m, and the second electric field strength is between 75-150V / m.

10. The device according to claim 9, characterized in that The pulse electricity provided by the pulse applying unit is direct current with a frequency ranging from 1 Hz to 20 kHz and a duty cycle of not less than 50%.

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