Different surfaces alter gene expressions of iNOS and ARG1, NO secretion
To investigate the role of selected surfaces on polarization state of macrophages cultured cells from each experimental group were analyzed for ARG1 and iNOS gene expression and NO secretion. Our results demonstrated that the transcript level of ARG1 in the phage containing surfaces was higher than the control surface at 2 days. During the 7 days culture of cells, the gene expression of ARG1 was significantly upregulated only in the cells cultured on surface compared to control surface (Figure 3A ). In addition, iNOS gene expression was significantly upregulated in the containing and Gelatin surfaces compared to their control counterparts after 2 days. While, the expression of iNOS was significantly downregulated after 7 days in . The results of qRT-PCR for iNOS gene expression were confirmed by the results of NO production in the culture supernatant of macrophages from each group (Figure 3C ). For determining the M1/M2 balance of cultured macrophages, the ARG1/iNOS mRNA expression ratio was measured. There was a significant increase in the ratio of ARG1/iNOS mRNA.
Different surfaces alter cellular redox potential in macrophages
The redox potential of cultured macrophages was assessed using the determination of intracellular ROS production. Here, culturing of macrophages on surfaces dramatically decreased the intracellular ROS level as compared to control surface in both 2 and 7 days (Figure 4 ).
Different surfaces alter phagocytosis and efferocytosis of cultured primary macrophages
To define the functional effects of selected surfaces on macrophage phagocytosis and efferocytosis, we analyzed the uptake of labeled yeasts and apoptotic cells by cultured cells. In this study, the capacity of macrophages for internalization of yeasts and apoptotic cells was evaluated by both fluorescent microscopy and flow cytometry. We found significantly increased uptake of apoptotic cells by macrophages cultured on on the second and seventh days after culture. However, macrophages from control and gelatin surfaces exhibited increased level of phagocytic activity in two and seven days (Figure 5A, B ).
Discussion:
after implantation graft, Inflammation is initiated. Prolonged primary inflammation can lead to tissue damage which can prevent regeneration. The M2 macrophage (or anti-inflammatory macrophages) has been shown to lead to more reduce tissue damage and increased ability to regenerated (Brown, Ratner, Goodman, Amar, & Badylak, 2012) (Badylak et al., 2008). Bacteriophages can stimulate immune cells and modulate both innate and adaptive immune responses(Van Belleghem et al., 2019). Based on Bacteriophage-immune system interaction, the or production of modulation scaffold that has not been considered. In The present study, we showed that the is biomaterial can modulate macrophage responses. For this purpose, we examined nteraction with macrophages as a representative of tissue resident immune cells in vitro. we showed change macrophage response and alter cytokine profile and polarization.
According to immunofluorescence and scanning electron microscopy analysis, the most dramatic increase was observed in the cell number and contact areas of cultured macrophages on structural support for adhesion that facilitate more cell proliferation and migration(X. Li, Dai, Shen, & Gao, 2017; Yang et al., 2017) (Richbourg, Peppas, & Sikavitsas, 2019). Previous reports demonstrated that RGD peptide plays a critical role in the spread of cells through focal adhesion [46]. In addition, our findings indicated that the combination with RGD peptide are both non-toxic and biocompatible.
Since macrophages are one of the key regulators of tissue remodeling, their response on candidate scaffolds may be an important indicator of successful tissue engineering. It has been well documented that macrophages contribute to modulate immune responses through their paracrine secretion (Corliss, Azimi, Munson, Peirce, & Murfee, 2016). During the early stages of normal wound healing, M1 macrophages infiltrate the wound to promote inflammation and to stimulate the wound healing process. M2 macrophages begin to accumulate around the third or fourth day after injury, while the level of M1 macrophages decreases. In tissue remodeling processes, M2 macrophages generate in several ways including direct shift of M1 type to M2 type macrophages, polarization of newly migrating macrophages toward M2 phenotype, and proliferation of other M2 macrophages (Yu, Tutwiler, & Spiller, 2015). In this study, we showed that the especially when combined with RGD, had the ability to reprogram naive peritoneal macrophages toward M2-like phenotype. Additionally, our results demonstrated that stimulate the secretion of TNF-α, IL-6, TGF- β and IL-10 at 2 days after culture. Also, we observed a time-dependent increase in M2 macrophage markers (IL-10 and TGF- β (Makita, Hizukuri, Yamashiro, Murakawa, & Hayashi, 2015)) and decrease in M1 macrophage marker (TNF-α (Shapouri‐Moghaddam et al., 2018)). In addition, cytokine analysis at both gene expression and protein level showed polarization of macrophages to M2 phenotype after interaction.
Moreover, M2 macrophages are metabolically different from M1 macrophages and the metabolic patterns of each are directly related to their tissue modulating function. It has been accepted that the Inducible nitric oxide synthase (iNOS) gene and NO are highly expressed in M1 macrophages, while the upregulation of arginase1 (ARG1) was observed in M2 macrophages (Jin, Liu, & Nelin, 2015).Notably, L-arginine metabolic pathway of macrophages determines the polarization status toward M1 or M2 phenotype (Rath, Müller, Kropf, Closs, & Munder, 2014). It has been widely accepted that NO is a key molecule produced by M1 macrophages to exert their role in immune defense. High level production of NO is generated from the oxidation of L-arginine. ARG1 is responsible for another metabolic pathway for L-arginine in macrophages, and this pathway produces L-ornithine for the biosynthesis of polyamine and collagen. These products help M2 macrophages to improve tissue regeneration (Z. Li et al., 2012; Rőszer, 2015). It is noteworthy to mention that the competition between ARG1 and NOS enzymes determine the M1/M2 phenotypic shift in macrophages. We found that the ratio of ARG1/iNOS transcript levels in the
It has been proposed that redox potential can play a complex role in the determination of macrophage cellular fate (Tan et al., 2016). A previous study demonstrated that ROS level is closely related to essential signaling pathways, which regulate M1 macrophage polarization (Lee et al., 2014). It is interesting to note that interactions between ROS and NO are responsible for the regulation of cellular inflammatory conditions (McNeill et al., 2015).
Furthermore, macrophages are responsible for the clearance of microbial pathogens (phagocytosis) and apoptotic cells (efferocytosis) (Korns, Frasch, Fernandez-Boyanapalli, Henson, & Bratton, 2011). Therefore, to ask if selected surfaces in the current study could alter the Functional properties of macrophages, we analyzed the phagocytic and efferocytic activity of each experimental group. In particular, proper efferocytosis by macrophages is an essential step in the regulation of tissue remodeling. With regard to this, alternatively activated M2 macrophages have enhanced efferocytic capability and therefore, contributed to the tissue regeneration process. In contrast, M1 macrophages are known to have low efferocytic properties (Korns et al., 2011; Sun et al., 2015).
Conclusion:
Biomaterials are most important part of strategies for regenerative medicine, In the process of tissue regeneration, modulation of immune and reduce anti-inflammatory responses are one of the most essential strategies to achieve desired tissue remodeling responses. Here, we demonstrated that s toward regenerative M2-like macrophages, promoting tissue remodeling especially through their secreted factors. effectively than In conclusion, we believe that the can be introduced as Immuno-modulating biomaterial for successful tissue regeneration and open new avenues for future in vivo tissue engineering studies.

## Conflict of interest

All the Authors declare no conflict of interest.
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