Abstract: BACKGROUND: The regulation of M1/M2 polarization direction of macrophages is particularly critical in tissue engineering applications, and timely regulation can minimize proinflammatory, anti-inflammatory, or tissue healing responses.
OBJECTIVE: To implant lentivirus-mediated miRNA-378a macrophage strain complex collagen to detect the expression level of immune regulation in the in vivo environment, and further clarify the influence of miRNA-378a in promoting macrophage M2 polarization in immune regulation and tissue repair in the in vivo environment.
METHODS: Lentivirus-mediated miRNA-378a overexpressing macrophage cell lines and negative control virus macrophage lines were amplified and screened, and the macrophage lines were recovered and cultured together with collagen sponge to form a composite scaffold, which was divided into the following groups: (1) Positive group: miRNA-378a overexpressing macrophage-collagen sponge composite; (2) negative group: negative control of virus-mediated miRNA-378a macrophage-collagen sponge composite; (3) control group: macrophage-collagen sponge; (4) blank control group: collagen sponge. The cell density, phenotype, and adhesion of each group were observed by immunofluorescence and scanning electron microscopy. The cells were implanted into the subcutaneous model of the back of mice, and the mice were sacrificed 4 and 7 days after modeling. The direction of macrophage polarization in the collagen sponge composite of macrophages with miRNA-378a overexpression mediated by lentivirus and its effect on immune regulation and tissue repair were analyzed by gross observation, hematoxylin-eosin staining, MASSON staining, and immunohistochemistry.
RESULTS AND CONCLUSION: (1) Under immunofluorescence microscopy, the macrophage cell lines in each group were observed to form a composite scaffold with the collagen sponge. (2) Under scanning electron microscope, lentivirus-mediated miRNA-378a macrophages in the positive group proliferated in cell density, had spherical, elliptic and polygonal differentiation, and had more pseudofeet than other groups. (3) Under general observation, the overall 7-day healing was better than that at 4 days. Lentivirus-mediated miRNA-378a macrophages in the positive group healed better than other groups regardless of 4 and 7 days. (4) Lentivirus-mediated miRNA-378a macrophages in the positive group under hematoxylin-eosin staining and MASSON staining had more amounts of fibrocytes, capillaries, fibroblasts, and collagen fiber hyperplasia. (5) Immunohistochemistry showed that lentivirus-mediated miRNA-378a macrophages in the positive group were more positive in 4- and 7-day M2 polarized cells than in other groups. The macrophages of the control and negative groups in 4- and 7-day M2 polarized cells were greater than that of the blank control group. There was no statistical difference between the control group and the negative group. The number of stained cells in the positive, negative, and control groups regardless of 4 and 7 days was higher than that in the blank control group, and the positive group > negative group ≈ control group > blank control group. (6) It is concluded that macrophages with miRNA-378a overexpression have a large amount of fibroblasts, capillaries, fibroblasts, and collagen fiber hyperplasia in vivo, which has a positive effect on tissue repair, and can promote the polarization of macrophages towards M2 type and inhibit the polarization of M1 type, thus contributing to reducing the inflammatory response of the body. 

Key words: miRNA-378a, lentiviral transfection, macrophage polarization, immunomodulation, tissue engineering