A method for culturing human periodontal ligament stem cells using self-assembling mussel myoglobin

Abstract

The application discloses a method for culturing human periodontal ligament stem cells by using self-assembled mussel myoglobin. The method comprises the following steps: dissolving the self-assembled mussel myoglobin in PBS to adjust the pH to 7.0-8.0, and filtering and sterilizing; adding the self-assembled mussel myoglobin into a cell culture medium to prepare a working culture medium with a final concentration of 10-100 mu g / mL; resuspending the cells after digesting hPDLSCs, and adjusting the density to 3.0*10<5>-3.5*10<5> per mL; using the hanging drop culture method, 20-30 mu L of each drop is inoculated in the inner side of a culture dish cover, and human periodontal ligament stem cells are obtained after 2-5 days of culture. The wet adhesion characteristics of the self-assembled mussel myoglobin are used to significantly improve the structural stability of stem cell spheres and the internal cell survival rate, and to enhance the stemness maintenance, paracrine function, immunoregulatory capacity and multi-directional differentiation potential of stem cells. The cell spheres prepared by the method can be used for tissue repair, and especially show excellent repair effect in the treatment of thin endometrium.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and regenerative medicine technology, specifically, it relates to a method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein. Background Technology

[0002] Stem cell therapy is a cutting-edge technology in regenerative medicine, with broad application prospects in tissue damage repair. Compared to traditional two-dimensional monolayer culture, three-dimensional cultured stem cell spheres can better simulate the in vivo cellular microenvironment, reduce epigenetic drift of stem cells, and maintain their stemness and functional activity, making it one of the key technologies for improving the efficacy of stem cell therapy. However, three-dimensional cultured stem cell spheres face significant technical bottlenecks: when the sphere diameter exceeds 200 μm, the transport distance for nutrients and oxygen to the interior of the sphere increases, leading to a hypoxic and nutrient-deficient environment in the core region, inducing apoptosis or necrosis; simultaneously, nutrient deficiency inhibits cellular metabolic activity, resulting in insufficient secretion of extracellular matrix, causing the sphere structure to loosen or even disintegrate, severely affecting the therapeutic effect after transplantation.

[0003] Human periodontal ligament stem cells (hPDLSCs) are a type of adult mesenchymal stem cells that reside within the periodontal ligament tissue. They possess self-renewal capacity and multi-lineage differentiation potential, capable of differentiating into various functional cell types such as fibroblasts, myofibroblasts, and neuron-like cells. Previous studies have shown that hPDLSCs have the potential to promote angiogenesis, immune regulation, and tissue repair. However, the three-dimensional culture of hPDLSCs also faces the aforementioned problems of structural instability and susceptibility to internal necrosis, limiting their application in regenerative medicine.

[0004] Mussel foot proteins (Mfps) are natural adhesion proteins secreted by marine mussels. Their core adhesion domain contains numerous dopa (DOPA) residues, enabling them to achieve strong adhesion in moist environments through hydrogen bonding, hydrophobic interactions, and metal coordination bonds, while also exhibiting excellent biocompatibility and degradability. Our laboratory previously prepared self-assembled mussel foot proteins using microbial engineering techniques, targeting the core adhesion domain (type) of mussel foot proteins (patent application number: CN202410397972.2). In vitro experiments confirmed their excellent wet adhesion ability and cell compatibility. However, there are currently no reports on using self-assembled mussel foot proteins for three-dimensional culture of hPDLSCs or on enhancing the structural stability and biological function of stem cell spheres through their adhesion properties. Summary of the Invention

[0005] The purpose of this invention is to provide a method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein, in order to solve the problems of unstable stem cell sphere structure, easy necrosis of internal cells, and low functional activity in existing three-dimensional culture technology.

[0006] The technical solution of the present invention is as follows: A method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive proteins includes the following steps: Step 1: Dissolve the self-assembled mussel adhesive protein (YV-cMfp) in PBS buffer, adjust the pH to 7.0-8.0, filter to sterilize, and prepare the self-assembled mussel adhesive protein stock solution; Step 2: Add the self-assembled mussel adhesive protein stock solution to the cell culture medium to prepare a working culture medium containing self-assembled mussel adhesive protein, wherein the final concentration of self-assembled mussel adhesive protein is 10-100 μg / mL. Step 3: Take human periodontal ligament stem cells in the logarithmic growth phase, digest and centrifuge them, then resuspend the cells in the working medium containing self-assembled mussel adhesive protein prepared in Step 2, and adjust the cell density to 3.0 × 10⁻⁶. 5 -3.5×10 5 cells / mL; Step 4: Using the hanging drop culture method, seed the cell suspension from Step 3 into the inside of the culture dish lid at a volume of 20-30 μL per drop, with each drop containing 6000-10000 cells. Add PBS to the bottom of the culture dish to maintain humidity and incubate in a 37°C, 5% CO2 incubator. Step 5: Culture for 2-5 days to obtain human periodontal ligament stem cells (hPDLSCs@YV-cMfp).

[0007] Preferably, in the above-described method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein, the final concentration of the self-assembled mussel adhesive protein in step 2 is 50 μg / mL.

[0008] Preferably, in the above-described method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein, the cell density in step 3 is 3.2 × 10⁻⁶. 5 Cells / mL; the volume of each drop in step 4 is 25 μL, and each drop contains 8000 cells.

[0009] Preferably, in the above-described method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein, the culture time in step 5 is 5 days.

[0010] Preferably, in the above-described method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein, the self-assembled mussel adhesive protein in step 1 is described in Chinese invention patent application number 2024103979722, and is the sequence described in SEQ ID NO. 4 of that patent.

[0011] The above-prepared human periodontal ligament stem cells are used in the preparation of tissue repair agents. The tissue repair includes endometrial repair, angiogenesis promotion, or inflammation regulation.

[0012] The functional identification of hPDLSCs@YV-cMfp cell spheroids obtained in step 5 includes: observing cell viability through live / dead staining, observing the surface morphology of the cell spheroids through scanning electron microscopy, observing the internal structure of the cell spheroids through HE staining, and detecting stem gene and cytokine expression through qPCR. The human periodontal ligament stem cell spheroids have a diameter of 200-250 μm, with tightly packed cells, clear cell boundaries, no obvious necrotic areas inside the spheroids, and a cell viability ≥90%. The expression of stem genes Oct4 and Sox2 in the cell spheroids is upregulated by 2-3 times or more, and the secretion levels of anti-inflammatory factor IL-10 and pro-angiogenic factors VEGF and PDGF-1 are upregulated by 1-2 times.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The culture method of this invention significantly improves the structural stability of stem cell spheres: Utilizing the wet adhesion properties of self-assembled mussel adhesive proteins, the adhesion between hPDLSCs cells is enhanced, allowing the cell spheres to maintain a complete and dense spherical structure even after 5 days of culture, avoiding structural loosening and cell detachment. Scanning electron microscopy results show that the human periodontal ligament stem cell spheres cultured by the method of this invention are tightly arranged, with complete individual cell outlines and clear boundaries; while the control group cell spheres exhibit a loose, dough-like appearance with blurred cell boundaries.

[0014] 2. Improved cell survival rate within stem cell spheres: The self-assembled mussel adhesive protein maintains the internal spatial structure of the cell spheres, providing a survival space for the internal cells and preventing cell death due to hypoxia. Live / dead staining and HE sectioning results showed that after 5 days of culture, the human periodontal ligament stem cells cultured using the method of this invention exhibited uniform nuclear staining and a significantly reduced number of necrotic cells; while the control group showed obvious necrotic areas and a large number of anucleate dead cells within the cell spheres.

[0015] 3. Enhanced stem cell stem cell maintenance: Compared with two-dimensional culture, the expression of stem cell genes Oct4 and Sox2 in human periodontal ligament stem cells cultured by the method of this invention was upregulated by more than 2-3 times after 5 days of culture (P<0.01), indicating that the self-assembled mussel adhesive protein and three-dimensional culture work synergistically to effectively maintain the self-renewal capacity and multi-directional differentiation potential of stem cells.

[0016] 4. Promotion of paracrine function: Compared with two-dimensional culture, the human periodontal ligament stem cells cultured by the method of this invention showed a 1-3 fold upregulation of gene expression levels of anti-inflammatory factor IL-10 and pro-angiogenic factors VEGF and PDGF-1 in the supernatant after 5 days of culture. In vitro functional verification showed that the supernatant could significantly accelerate lumen formation of human umbilical vein endothelial cells (lumen tendency appeared after 4 hours, and mature blood vessel-like structures were formed after 6 hours), and the number of lumen nodes and blood vessel length were significantly higher than those in the control group (P<0.001).

[0017] 5. Broad application prospects: The human periodontal ligament stem cells prepared by the method of this invention can withstand mechanical forces (such as injection). In the mouse model of thin endometrium treated by in situ injection into the uterus, they can significantly promote the morphological repair and functional recovery of the endometrium, restore the endometrial thickness to the normal level, restore the number of glands, distribute collagen evenly, and enhance angiogenesis. This provides a safe and effective new strategy for the treatment of tissue damage diseases such as thin endometrium. Attached Figure Description

[0018] Figure 1 The image shows the detection results of hPDLSCs treated with different concentrations of self-assembled mussel adhesive protein. A is a microscopic image. B is the MTT cytotoxicity assay result. The results show normal cell viability within the concentration range of 10-100 μg / mL, with a slight proliferative effect at 50 μg / mL.

[0019] Figure 2 Bright-field images of the three-dimensional spherical morphology of hPDLSCs on day 2 and day 5, showing the effect of self-assembled mussel adhesive proteins. The images reveal that the spherical structures in the control group were loose on day 5, while those in the self-assembled mussel adhesive protein treatment group were intact and dense.

[0020] Figure 3 The images show the results of live and dead staining of hPDLSCs. A shows direct staining of cell spheres; B shows staining after cell spheres are dispersed. The results indicate that the survival rate of the self-assembled mussel adhesive protein treatment group was significantly higher than that of the control group on day 5.

[0021] Figure 4 The image shows a scanning electron microscope image of hPDLSCs. It reveals that the cells in the self-assembled mussel adhesive protein treatment group are tightly packed with clear boundaries, while the control group has loose surfaces and blurred boundaries.

[0022] Figure 5 This is an HE-stained section of hPDLSCs. It shows that the cell nuclei in the self-assembled mussel adhesive protein treatment group are uniformly stained and have less necrosis, while the control group shows obvious necrotic areas.

[0023] Figure 6 This study investigated the changes in stem gene expression levels of hPDLSCs after two-dimensional and three-dimensional co-culture of self-assembled mussel adhesive proteins with hPDLSCs.

[0024] Figure 7 This image shows the effect of hPDLSCs on thin endometrial tissue in mice. In the hPDLSCs ball treatment group, the blue areas increased, but their distribution was irregular. In the hPDLSCs@YV-cMfp group, the blue areas decreased compared to the control group, and were widely distributed between the myometrium and the basal layer of the endometrium. The hPDLSCs@YV-cMfp treatment group showed extensive blue area distribution, uniform collagen distribution, and collagen deposition levels closest to the normal group. The tissue repair effect of hPDLSCs@YV-cMfp (50 μg / mL) was better than that of 10 μg / mL, and it was similar to the tissue state of a normal uterus. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] Example 1: Preparation and formulation of self-assembled mussel adhesive protein Take 2 mg of irradiated self-assembled mussel adhesive protein (prepared according to the method described in patent CN202410397972.2) and accurately weigh it using an electronic balance. Resuspend the protein in PBS buffer to a final concentration of 1 mg / mL. Add an appropriate amount of NaOH solution (0.1 M) to aid dissolution until the protein is completely dissolved. Filter the solution through a 0.22 μm filter for sterilization. Adjust the pH of the solution to 7.0-8.0. Add the above protein solution to DMEM high-glucose medium containing 10% fetal bovine serum according to experimental requirements to prepare working media of self-assembled mussel adhesive protein with final concentrations of 10 μg / mL, 30 μg / mL, 50 μg / mL, and 100 μg / mL. Store at 4°C in the dark for later use.

[0027] Example 2: Detection of cytotoxicity of self-assembled mussel adhesive proteins against hPDLSCs hPDLSCs in good growth condition (P3-P6 generations) were selected, and the cells were digested with 0.25% trypsin (containing EDTA). The cell pellet was collected by centrifugation at 1200 rpm for 5 min. The cells were resuspended in DMEM medium containing 10% FBS, and the cell suspension concentration was adjusted to 3 × 10⁻⁶. 4 100 μL per well (3 × 10⁶ cells / mL). 3Cells were seeded into 96-well cell culture plates and incubated overnight at 37°C, 5% CO2, and saturated humidity. When cell confluence reached 80%-90%, the culture medium was replaced with 2% serum DMEM containing 10, 30, 50, and 100 μg / mL self-assembled mussel adhesive protein, while the control group received 2% fetal bovine serum medium without self-assembled mussel adhesive protein. On days 2 and 5, 10 μL of MTT solution (final concentration 1 mg / mL) was added to each well, and incubation continued for 4 hours. The supernatant was discarded, and 100 μL of DMSO was added to each well, gently agitated to completely dissolve the formazan crystals. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader, and cell viability was calculated.

[0028] The results are as follows Figure 1 As shown, within the concentration range of 10-100 μg / mL, the survival rate of hPDLSCs remained at a normal level, and no obvious toxic reactions were observed. At a concentration of 50 μg / mL, cell proliferation activity was slightly increased, exhibiting a mild proliferative effect. Microscopic observation revealed black granules (protein precipitation) on and around the cells in the 100 μg / mL group after 2 days of culture. Therefore, subsequent experiments used 10, 30, and 50 μg / mL as the drug administration concentrations.

[0029] Example 3: Hanging drop culture of human periodontal ligament stem cells Collect the old culture medium from the hPDLSC culture dishes, add 500 μL of 0.25% EDTA-trypsin, incubate at 37℃ for 30 s, then terminate digestion with the old culture medium and detach the cells; centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend the cells in 1 mL of DMEM high-glucose medium containing 10% FBS; dilute the cell suspension according to the group requirements (control group, 10 μg / mL protein group, 30 μg / mL protein group, 50 μg / mL protein group): the hanging drop culture parameters are 70 beads / dish, 8000 cells per bead (volume 25 μL), and the final cell concentration is adjusted to 3.2 × 10⁻⁶ cells / dish. 5 Cells / mL; add 5mL PBS to the bottom of the culture dish to maintain humidity; perform hanging drop culture according to the above grouping, observe the morphology of cell spheroids daily, and take pictures and record them on the 2nd and 5th days after spheroidization.

[0030] The results are as follows Figure 2 As shown, on day 2 of culture, hPDLSCs in all groups were able to successfully aggregate to form stem cell spheres with no significant difference in sphere morphology. On day 5 of culture, the stem cell spheres in the control group showed obvious structural loosening, with a large number of detached single cells visible around the spheres. In contrast, the stem cell spheres in the 10, 30, and 50 μg / mL self-assembled mussel adhesive protein treatment groups maintained an intact and dense spherical structure without significant cell dispersion.

[0031] Example 4: Cell viability detection of human periodontal ligament stem cells by staining 4.1 Direct staining of cell spheres (live or dead) Collect hPDLSCs cell spheres cultured for 2 and 5 days and transfer them to EP tubes; centrifuge at 1200 rpm for 5 min to precipitate the cell spheres; gently wash once with PBS; add 1 μL of AO and 10 μL of PI to 1 mL of dilution buffer and mix well; add 200 μL of staining solution to each tube and incubate in the dark for 10 min; after staining, centrifuge, add 100 μL of PBS to resuspend the cell spheres, transfer to 48-well plates, and take pictures under a fluorescence microscope.

[0032] 4.2 Live and dead cell staining after cell spheroid dispersion Collect cell spheroids, resuspend them in 300 μL of 0.25% EDTA-trypsin, transfer them to 48-well plates for digestion, gently pipetting every 30 s and observing the digestion under a microscope; after the cells are completely dispersed, stop the digestion with 300 μL of culture medium and centrifuge at 1200 rpm for 5 min; wash with PBS and perform AO / PI staining as described above.

[0033] The results are as follows Figure 3 As shown, no obvious cell death signals were observed in any group on day 2 of culture; on day 5 of culture, obvious cell death signals appeared in the control group, while the self-assembled mussel adhesive protein treatment group maintained a high survival rate. Staining results after cell sphere dispersion showed that the proportion of dead cells in the control group was significantly increased, while only a small number of dead cells appeared in the self-assembled mussel adhesive protein treatment group, confirming that self-assembled mussel adhesive protein can significantly improve the survival rate of hPDLSCs spheres during in vitro 3D culture.

[0034] Example 5: Scanning electron microscopy observation of human periodontal ligament stem cells hPDLSCs cell spheres cultured for 2 and 5 days were collected and fixed overnight at 4°C with 4% paraformaldehyde. After washing with PBS, the cells were dehydrated using an alcohol gradient dehydration method (50%, 60%, 70%, 80%, 90%, 100% ethanol solution, 15 min each time) and air-dried. After gold sputtering, the surface structure of the stem cell spheres was observed under a scanning electron microscope.

[0035] The results are as follows Figure 4 As shown, the control group stem cell spheres exhibited a loose, dough-like appearance with blurred cell boundaries, making it difficult to distinguish the independent morphology of individual cells. The sphere diameter was approximately 260 μm. In contrast, the self-assembled mussel adhesive protein treatment group showed clearly spherical, tightly packed single cells with intact outlines and clear boundaries. The diameter of the spheres was approximately 220 μm on day 2 and approximately 240 μm on day 5. These results indicate that the self-assembled mussel adhesive protein makes the cell sphere structure more compact.

[0036] Example 6: HE staining analysis of human periodontal ligament stem cells Human periodontal ligament stem cells cultured for 2 and 5 days were collected and fixed overnight in 4% paraformaldehyde. After washing with PBS, the cells were coated with pre-warmed and melted 2% agarose solution. The cells were then subjected to gradient dehydration treatment (75%, 85%, 90%, 95% ethanol, anhydrous ethanol, benzene, xylene) and embedded in paraffin. After sectioning, the cells were stained with hematoxylin and eosin (HE) and observed under an optical microscope.

[0037] The results are as follows Figure 5 As shown, inside the hPDLSCs spheroids, individual cells are spindle-shaped or fusiform, and there are a large number of nucleated necrotic cells inside the spheroids; while inside the human periodontal ligament stem cell group, individual cells are round, the nuclei are uniformly stained, and the number of necrotic cells is significantly reduced, consistent with the results of live and dead cell staining.

[0038] Example 7: Detection of changes in the expression levels of stem genes, anti-inflammatory factors, and pro-angiogenic factors in hPDLSCs@YV-cMfp by quantitative real-time PCR In both 2D and 3D culture systems, after 5 days of culture, hPDLSCs@YV-cMfp were digested with trypsin, and the digestion was terminated. The cells were centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. After washing once with PBS, the cell pellet was collected by centrifugation (cell groups: control group, 10 μg / mL, 30 μg / mL, and 50 μg / mL LYV-cMfp groups). Total RNA was extracted from the cells according to the RNA extraction kit instructions. RNA concentration and purity (OD260 / 280 ratio) were measured using a nucleic acid detector. cDNA synthesis and qPCR were performed using a reverse transcription system: the qPCR reaction system included SYBR GreenMix, forward and reverse primers, cDNA template, and enzyme-free water. Reaction mixtures were prepared sequentially according to the experimental groups. Amplification was performed using a real-time quantitative PCR instrument. After the experiment, the relative expression level of the target gene was calculated using the 2^-ΔΔCt method, and statistical analysis was performed.

[0039] The results are as follows Figure 6 The results showed that under the 3D culture system, the expression of stem genes Oct4 and Sox2 in hPDLSCs@YV-cMfp cells was upregulated by more than 2-3 times on day 5, and the secretion levels of anti-inflammatory factor IL-10 and pro-angiogenic factors VEGF and PDGF-1 were upregulated by more than 1-3 times.

[0040] Example 8: Human periodontal ligament stem cell therapy for thin endometrium in mice Mice in estrus were anesthetized with isoflurane, their abdomens were shaved and disinfected, and the uterus was exposed by incision along the midline of the abdomen. A Y-shaped bifurcation at one uterine horn was bound with 5-0 surgical sutures. 50-80 μL of 95% alcohol was injected into an insulin needle above the suture, left for 30 seconds, and then aspirated. The uterus was washed 2-3 times with PBS. The sutures were cut, the uterus was repositioned, and the muscle and epidermal layers were sutured sequentially. On day 3 of modeling, the uterus appeared red and adhered. On day 7, the uterus was distended, and the endometrial thickness decreased to 151±6.0 μm, significantly lower than the normal group's 387±5.8 μm (P<0.001), indicating successful model establishment.

[0041] Mice were divided into 5 groups: normal group, model group, hPDLSCs sphere group, and hPDLSCs@YV-cMfp sphere group (10 μg / mL and 50 μg / mL). After modeling, the treatment groups received in-situ injections of the corresponding stem cell spheres (60 spheres / injection) into the uterus via insulin injection. Mice were euthanized 14 days after treatment, and the uterus was harvested and stained with Masson's stain. Figure 7 In the hPDLSCs@YV-cMfp ball treatment group, collagen distribution was uniform, similar to the normal group; in the model group, collagen was significantly lost. These results indicate that hPDLSCs@YV-cMfp balls can effectively promote the morphological repair and functional recovery of thin endometrium in mice.

Claims

1. A method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein, characterized in that, Includes the following steps: Step 1: Dissolve the self-assembled mussel adhesive protein in PBS buffer, adjust the pH to 7.0-8.0, filter to sterilize, and prepare a stock solution of the self-assembled mussel adhesive protein; Step 2: Add the self-assembled mussel adhesive protein stock solution to the cell culture medium to prepare a working culture medium containing self-assembled mussel adhesive protein, wherein the final concentration of self-assembled mussel adhesive protein is 10-100 μg / mL. Step 3: Human periodontal ligament stem cells (hPDLSCs) in the logarithmic growth phase were harvested, digested, and centrifuged. The cells were then resuspended in the working medium containing self-assembled mussel adhesive proteins prepared in Step 2, and the cell density was adjusted to 3.0 × 10⁻⁶ cells / year. 5 -3.5×10 5 cells / mL; Step 4: Using the hanging drop culture method, seed the cell suspension from Step 3 into the inside of the culture dish lid at a volume of 20-30 μL per drop, with each drop containing 6000-10000 cells. Add PBS to the bottom of the culture dish to maintain humidity and incubate in a 37°C, 5% CO2 incubator. Step 5: Culture for 2-5 days to obtain human periodontal ligament stem cells.

2. The method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein according to claim 1, characterized in that, The final concentration of the self-assembled mussel adhesive protein described in step 2 is 50 μg / mL.

3. The method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein according to claim 1, characterized in that, The cell density mentioned in step 3 is 3.2 × 10⁻⁶. 5 Cells / mL; the volume of each drop in step 4 is 25 μL, and each drop contains 8000 cells.

4. The method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein according to claim 1, characterized in that, The cultivation time described in step 5 is 5 days.

5. The method for culturing human periodontal ligament stem cells using self-assembled mussel adhesive protein according to claim 1, characterized in that, The self-assembled mussel adhesive protein described in step 1 is recorded in Chinese invention patent application number 2024103979722, and is the sequence described in SEQ ID NO. 4 of that patent.

6. The use of the human periodontal ligament stem cells prepared according to claim 1 in the preparation of tissue repair agents.

7. The application according to claim 6, characterized in that, The tissue repair includes endometrial repair, angiogenesis promotion, or inflammation regulation.

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