Mechanical and biochemical coupling regulation and control method and stem cell spatial differentiation system

Abstract

The invention provides a mechanical and biochemical coupling regulation and control method and a stem cell spatial differentiation system, and relates to the technical field of regenerative medicine and tissue repair, the mechanical and biochemical coupling regulation and control method comprises the following steps: S1, applying 1 Hz + / -0.2 Hz and 10% + / -0.5% periodic compression strain, activating YAP / TAZ nuclear transposition through an integrin beta1-FAK pathway, the nuclear localization proportion of YAP / TAZ being greater than 65%; s2, triggering a BMP-2 secretion gradient when the YAP nuclear localization proportion is greater than 70%, and S3, establishing a bidirectional biochemical gradient through a microfluidic system: pouring TGF-beta1 into a core region to be 20ng / mL, pouring dexamethasone into a marginal region to be 50ng / mL, and adding SDF-1alpha into the marginal region to be 50ng / mL. Preferably, the chip of the micro-fluidic gradient module is designed into a tree-shaped fractal channel, and the gradient half-decay distance is 300 microns. Preferably, the inoculation density of the dental pulp stem cells is 5 * 10 cells / cm < 2 >. According to the invention, the YAP / TAZ pathway is regulated and controlled through mechanical-biochemical coupling, the spatial differentiation of stem cells is realized, the resolution ratio reaches 200 microns, the construction period is shortened by 50%, and the transplantation success rate is greater than 90%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of regenerative medicine and tissue repair technology, in particular to a mechanical and biochemical coupling regulation method and a stem cell spatial differentiation system. BACKGROUND

[0002] The present application relates to the field of regenerative medicine and tissue repair technology, in particular to a mechanical and biochemical coupling regulation method and a stem cell spatial differentiation system.

[0003] In view of the above-mentioned related content, the following technical defects exist: the existing stem cell differentiation regulation technology has the following significant limitations: 1. Mechanical stimulation is mostly activated by a single path, resulting in insufficient osteogenic differentiation efficiency; 2. The spatial resolution of the biochemical gradient system is low, and the osteogenic / angiogenic area overlap is > 35%; 3. There is a lack of dynamic coupling mechanism of mechanical-biochemical signals, which cannot realize real-time feedback regulation based on YAP nuclear localization. SUMMARY

[0004] The purpose of the present application is to solve the problems existing in the prior art, and a mechanical and biochemical coupling regulation method and a stem cell spatial differentiation system are provided.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] S1, a periodic compression strain of 1Hz±0.2Hz, 10%±0.5% is applied to activate YAP / TAZ nuclear translocation through integrin β1-FAK pathway, and the nuclear localization ratio of YAP / TAZ is greater than 65%;

[0007] S2, when the YAP nuclear localization ratio is > 70%, a BMP-2 secretion gradient is triggered,

[0008] S3, a two-way biochemical gradient is established by a microfluidic system: the core area is perfused with TGF-β1 at 20ng / mL, dexamethasone at 100nM, the edge area is perfused with VEGF at 50ng / mL, SDF-1α is added every 30ng / mL, and the spatial decay coefficients of osteogenic and angiogenic signals are ≥0.15 / mm≥0.12 / mmR 2 ≥0.95.

[0009] Preferably, the real-time monitoring impedance spectrum of the mechanical loading is Δθ>8° at 1kHz frequency and the oxygen partial pressure gradient is 5%→21% O.

[0010] Preferably, the YAP / TAZ activation depends on the integrin β1-FAK pathway, and the FAK inhibitor is 10μM PF-573228, which can reduce the phosphorylation level by > 90%.

[0011] Preferably, the mechanical loading module: frequency 1Hz±0.2Hz, strain amplitude 10%±0.5% compression strain generator, microfluidic gradient module: double-channel perfusion system, core zone flow rate 0.5mL / min, edge zone 1.5mL / min, dynamic monitoring module: impedance analyzer phase angle Δθ>8° and oxygen sensor 5%→21% O.

[0012] Preferably, the microfluidic gradient module chip is designed as a tree-shaped fractal channel, and the gradient half-life distance is 300μm.

[0013] Preferably, the dental pulp stem cell seeding density is 5×10 cells / cm 2 .

[0014] Preferably, the osteogenic differentiation marker ALP activity of the same reaches 38.5±3.2U / mg in the core zone, and the CD31+ blood vessel-like structure density in the edge zone is 21±3 / mm 2 .

[0015] Preferably, the trabecular bone spacing is 152±18μm, and the vascular density difference from the natural tissue is <12%.

[0016] Preferably, the construction cycle is ≤14 days, and the functional integration success rate after transplantation for 6 months is >90%.

[0017] Preferably, the FAK inhibitor is used in blocking mechanical signal conduction, and the inhibition rate is >90%.

[0018] Compared with the prior art, the application has the advantages and positive effects that,

[0019] 1、In the application, the YAP / TAZ pathway is regulated by mechanical-biochemical coupling, the spatial differentiation of stem cells is realized, the resolution reaches 200μm, the construction cycle is shortened by 50%, and the transplantation success rate is >90%. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a schematic diagram of the YAP / TAZ signal pathway activation mechanism of the application;

[0021] Figure 2 It is a schematic diagram of the microfluidic gradient generation system of the application;

[0022] Figure 3 It is a schematic diagram of the spatial differentiation effect of the application. DETAILED DESCRIPTION

[0023] In order to enable the above-mentioned objects, features and advantages of the present application to be more clearly understood, the following will further describe the present application with reference to the accompanying drawings and examples. It should be noted that the examples of the present application and the features in the examples can be combined with each other without conflict.

[0024] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced without the specific details. In other instances, well-known methods have not been described in detail in order not to unnecessarily obscure aspects of the present application.

[0025] Example 1, refer to Figure 1 As shown in the figure, the present application provides a mechanical and biochemical coupling regulation method and a stem cell spatial differentiation system, comprising the following steps:

[0026] S1, a periodic compression strain of 1Hz±0.2Hz, 10%±0.5% is applied to activate YAP / TAZ nuclear translocation through integrin β1-FAK pathway, and the nuclear localization ratio of YAP / TAZ is greater than 65%;

[0027] S2, when the nuclear localization ratio of YAP is greater than 70%, BMP-2 secretion gradient is triggered,

[0028] S3, a bidirectional biochemical gradient is established by a microfluidic system: TGF-β1 of 20ng / mL and dexamethasone of 100nM are perfused in the core area, VEGF of 50ng / mL and SDF-1α of 30ng / mL are added in the edge area, and the spatial attenuation coefficients of osteogenic and angiogenic signals are greater than or equal to 0.15 / mm and 0.12 / mm, respectively. 2 The real-time monitoring impedance spectrum of mechanical loading is greater than 8° at 1kHz frequency, and the oxygen partial pressure gradient is 5%→21% O.

[0029] Example 2, refer to Figure 2 As shown in the figure, YAP / TAZ activation depends on integrin β1-FAK pathway, and the FAK inhibitor is 10μM PF-573228, which can reduce the phosphorylation level by more than 90%. The mechanical loading module is a compression strain generator with a frequency of 1Hz±0.2Hz and a strain amplitude of 10%±0.5%. The microfluidic gradient module is a double-channel perfusion system with a core area flow rate of 0.5mL / min and an edge area flow rate of 1.5mL / min. The dynamic monitoring module is an impedance analyzer with a phase angle Δθ greater than 8° and an oxygen sensor with an oxygen partial pressure gradient of 5%→21% O. The chip design of the microfluidic gradient module is a tree-shaped fractal channel with a gradient half-decay distance of 300μm. The dental pulp stem cell seeding density is 5×10 2 The osteogenic differentiation marker ALP activity in the core area is 38.5±3.2U / mg, and the CD31+vascular-like structure density in the edge area is 21±3 / mm 2, trabecular bone spacing 152 ± 18 μm, blood vessel density difference < 12% from natural tissue, construction cycle ≤ 14 days, functional integration success rate > 90% after 6 months of transplantation, application of FAK inhibitors in blocking mechanical signal transduction, inhibition rate > 90%.

[0030] Example 3, refer to Figure 3 As shown in the figure, Western Blot shows that the expression of YAP in the strain group is increased by 3.2 times, p<0.01, and the phosphorylation level of TAZ is increased by 4.1 times; the signal transduction in the FAK inhibitor group is completely blocked.

Claims

1. A mechanical and biochemical coupling regulation method and a stem cell spatial differentiation system, comprising the following steps: S1. Apply periodic compressive strain of 1Hz±0.2Hz and 10%±0.5% to activate YAP / TAZ nuclear translocation through the integrin β1-FAK pathway, wherein the nuclear localization ratio of YAP / TAZ is greater than 65%. When the S2 and YAP nuclear localization ratio is greater than 70%, the BMP-2 secretion gradient is triggered. S3. A bidirectional biochemical gradient was established using a microfluidic system: the core region was perfused with 20 ng / mL TGF-β1 and 100 nM dexamethasone, while the peripheral region was perfused with 50 ng / mL VEGF and 30 ng / mL SDF-1α. The spatial attenuation coefficients of osteogenic and angiogenic signal intensities were ≥0.15 / mm and ≥0.12 / mmR, respectively. 2 >0.

95.

2. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 1, characterized in that: The impedance spectrum for real-time monitoring of mechanical loading is Δθ > 8° and oxygen partial pressure gradient 5% → 21%O at a frequency of 1 kHz.

3. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 1, characterized in that: The YAP / TAZ activation depends on the integrin β1-FAK pathway. The FAK inhibitor is 10 μMPF-573228, which can reduce phosphorylation levels by >90%.

4. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 1, characterized in that: The mechanical loading module includes a compressive strain generator with a frequency of 1Hz±0.2Hz and a strain amplitude of 10%±0.5%. The microfluidic gradient module is a dual-channel infusion system with a flow rate of 0.5mL / min in the core area and 1.5mL / min in the edge area. The dynamic monitoring module includes an impedance analyzer with a phase angle Δθ>8° and an oxygen sensor with a flow rate of 5%→21%O.

5. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 1, characterized in that: The microfluidic gradient module is designed as a tree-like fractal channel with a gradient half-life distance of 300 μm.

6. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 1, characterized in that: The dental pulp stem cell seeding density was 5 × 10 cells / cm². 2 .

7. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 1, characterized in that: The osteogenic differentiation marker ALP activity reached 38.5±3.2 U / mg in the core region and the CD31+ vascular-like structure density in the peripheral region was 21±3 / mm. 2 .

8. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 1, characterized in that: Prepared by any one of claims 1-7, the trabecular spacing is 152±18μm, and the vascular density differs from that of natural tissue by <12%.

9. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 8, characterized in that: The construction cycle is ≤14 days, and the functional integration success rate is >90% 6 months after the transplant.

10. The mechanical and biochemical coupling regulation method and stem cell spatial differentiation system according to claim 8, characterized in that: The application of FAK inhibitors in blocking mechanical signal transduction shows an inhibition rate of >90%.

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