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Published in final edited form as: Appl Mater Today. 2019 Nov 26;18:100508. doi: 10.1016/j.apmt.2019.100508

ECM-mimicking nanofibrous matrix coaxes macrophages toward an anti-inflammatory phenotype: Cellular behaviors and transcriptome analysis

Rui-Xin Wu a,b, Chi Ma a, Yongxi Liang a, Fa-Ming Chen b,*, Xiaohua Liu a,**
PMCID: PMC7450828  NIHMSID: NIHMS1061552  PMID: 32864422

Abstract

An in-depth understanding of biomaterial cues to selectively polarize macrophages is beneficial in the design of “immuno-informed” biomaterials that positively interact with the immune system to dictate a favorable macrophage response following implantation. Given the promising future of ECM-mimicking nanofibrous biomaterials in biomedical application, it is essential to elucidate how their intrinsic cues, especially the nanofibrous architecture, affect macrophages. In the present study, we evaluated how the nanofibrous architecture of a gelatin matrix modulated macrophage responses from the perspectives of cellular behaviors and a transcriptome analysis. In our results, the nanofibrous surface attenuated M1 polarization and down-regulated the inflammatory responses of macrophages compared with a smooth surface. Besides, the cell-material interaction was up-regulated and the adhered macrophages tended to maintain an original, non-polarized state on the nanofibrous matrix. Accordingly, whole transcriptome analysis revealed that nanofibrous architecture up-regulated the pathways related to ECM-receptor interaction and down-regulated pathways related to pro-inflammation. This study provides a panoramic view of the interaction between macrophages and nanofibers, and offers valuable information for the design of immunomodulatory ECM-mimicking biomaterials for tissue regeneration.

Keywords: Macrophages, Extracellular matrix, Gelatin, Nanofibers, Inflammation, Adhesion, Transcriptome

Graphical Abstract

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1. Introduction

As an essential component of the tissue engineering triad, biomaterials not only provide mechanical support for cells but also serve as carriers for bioactive molecules (e.g. growth factors). With the advances in tissue engineering, a number of bio-inspired materials have been developed to manipulate stem cell behaviors, and some have shown promising outcomes in vitro [1–4]. However, when implanted in vivo, most of the biomaterials failed to function as expected, and some even led to acute or chronic inflammation [5]. These unsatisfying outcomes are largely due to an undesirable interaction between implanted biomaterials and the immune system of a host.

Macrophages are the critical immune regulators, and can either promote inflammation or facilitate repair and regeneration owing to their plasticity and versatility [6]. Based on the polarization states in response to environmental stimulus such as implanted biomaterials, macrophages can be roughly divided into a pro-inflammatory M1 phenotype and a pro-healing M2 phenotype [7]. In general, a higher M2/M1 ratio of macrophages surrounding the implanted biomaterials is associated with better remodeling outcomes [8]. However, excessive M2 response may also contribute to detrimental fibrosis [9, 10]. Therefore, identification of the intrinsic biomaterial characteristics that regulate macrophage phenotypes is pivotal to the development of novel immunomodulatory biomaterials for tissue repair and regeneration.

Biomaterials regulate macrophage phenotypes through appropriate biochemical cues (e.g. composition and surface chemistry) and biophysical cues (e.g. stiffness and surface topography) [6, 11, 12]. For instance, a lower molecular weight of hyaluronic acid was reported to be associated with M1-like inflammation, while a higher molecular weight of the hyaluronic acid was shown to be involved in M2-like activation [13]. Biomaterial stiffness was demonstrated to play a role in modulating macrophage behaviors as well [14, 15]. For example, macrophages cultured on soft polyacrylamide gels generated lower amounts of inflammatory cytokines than on stiff substrates after exposure to lipopolysaccharide and tumor necrosis factor-α [16]. Further work indicated that the substrate stiffness modulated macrophage plasticity through actin polymerization and Rho-GTPase activity [16]. The effects of biomaterial surface topography on macrophage polarization and phenotype have also been explored for over two decades. However, most of the work focused on examining how artificial surface patterns such as nano gratings, pillars, nods, or grooves modulate macrophage responses [17–21]. Consequently, our understanding of how macrophages interact with nanofibrous structure, which resembles the architecture of natural extracellular matrix (ECM), is limited [14, 22, 23]. To date, how the nanofibrous architecture modulates macrophage responses at the transcriptome level remains unknown.

Gelatin is a widely used natural biomaterial and is obtained by partial hydrolysis of collagen, the most abundant protein in native ECM [24]. Gelatin not only retains the superiority of collagen in terms of biocompatibility and biodegradability, but also possesses unique advantages such as non-immunogenicity and cost efficiency, making gelatin a popular biomaterial used in tissue regeneration. With the advances of material engineering techniques such as phase separation and electrospinning, gelatin can be readily processed into collagen-like nanofibrous matrix [25, 26]. Moreover, a gelatin based nanofibrous matrix mimics both the chemical compositions and physical architecture of natural ECM, and is an excellent substrate for cell adhesion, proliferation, differentiation, and neo-tissue regeneration [26–31].

In this study, we examined how the nanofibrous architecture of gelatin matrix modulates macrophage phenotypes. We first analyzed the responses of macrophages (RAW 264.7 cells) to nanofibrous and smooth architecture with respect to adhesion, morphology, and cytokine secretion. Next, a whole transcriptome analysis was performed separately to both the nanofibrous and smooth architecture to reveal differential gene expression profiles reflecting macrophage polarization and signaling pathway. This information provides an in-depth understanding of the interaction between nanofibrous architecture and macrophages.

2. Materials & Methods

2.1. Materials

Gelatin (from bovine skin, type B, 225 g Bloom), acetic acid, fluorescein isothiocyanate (FITC), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), glycine, and penicillin-streptomycin were purchased from Sigma-Aldrich (St. Louis, USA). TritonX-100, ethyl acetate, paraformaldehyde, and glutaraldehyde were bought from VWR (Radnor, USA). Hexafluoroisopropanol was acquired from Oakwood Chemical (Estill, USA). N-hydroxysuccinimide (NHS) and 4-morpholineethanesulfonic acid (MES) were obtained from Acros Organics (Geel, Belgium). Phosphate buffered saline (PBS), alpha-minimum essential medium (α-MEM), fetal bovine serum (FBS), and goat serum were secured from Gibco (Carlsbad, USA). Hoechst 33342 was purchased from Pierce Biotechnology (Waltham, USA). RNeasy Mini Kit was acquired from Qiagen (Hilden, Germany). SoAdvanced™ Universal SYBR® Green Supermix and iScript™ gDNA Clear cDNA Synthesis Kit were bought from BioRad (Hercules, USA). Anti-Mannose Receptor antibody ab64693 and Anti-iNOS antibody ab15323 were obtained from Abcam (Cambridge, UK). An Alexa Fluor Plus 555 secondary antibody A32732 and a ProLong™ Gold Antifade Mountant were purchased from Invitrogen (Carlsbad, USA). CF633 phalloidin was bought from Biotium (Fremont, USA). TruSeq®Stranded mRNA LT kit was purchased from Illumina (San Diego, USA). RAW 264.7 (the murine macrophage cell line) was obtained from ATCC (Manassas, USA). Sprague-Dawley rats were obtained from the Jackson laboratory (Bar Harbor, USA).

2.2. Preparation of gelatin matrices

Nanofibrous gelatin matrices were fabricated using an electrospinning process as previously described [26] with minor modification. Briefly, 1.6 g of gelatin and 0.004 g of FITC-conjugated gelatin prepared according to an established protocol [32] were dissolved in 10 mL of acetic acid/ethyl acetate/water/hexafluoroisopropanol (2.5:1.5:1:5) solvent mixture. A high voltage of 11.5 kV and a feeding rate of 0.5 mL/h were applied to fabricate gelatin nanofibers, which were then collected on the aluminum foil attached to a rotating drum with a rotating speed of 80 rpm. Smooth gelatin matrices were produced by casting 2% of an aqueous gelatin solution on glass Petri dishes and air drying the mixture under a fume hood. Next, both the nanofibrous and smooth gelatin matrices were crosslinked using an EDC/NHS crosslinking system as described in our previous study [33]. After this procedure, the gelatin matrices were soaked in a glycine solution for 24 h (8 g/L), washed with distilled water, dehydrated in ethanol, and vacuum-dried. Later, the matrices were sputter coated with gold and observed under a JSM6010 scanning electronic microscope (SEM) (JEOL, Japan). The diameters of the nanofibers were measured as described in our previous publications [25, 26] using the ImageJ software that is available for free from www.nih.gov.

2.3. Cell seeding on gelatin matrices

The matrices were rehydrated, cut into 25 mm × 25 mm squares, and fixed onto CellCrown™12NX (Scaffdex, Finland) devices. The assembled matrices were then disinfected in 75% ethyl alcohol for 8 h, washed with sterile deionized water and 1 × PBS three times, and soaked in α-MEM overnight. After that, the assembled matrices were inserted into 12-well culture plates that were prefilled with 2 mL complete culture medium containing α-MEM with 10% FBS and 1% penicillin-streptomycin. RAW 264.7 macrophages (1 × 105 cells) were seeded onto a gelatin matrix and cultured in 500 μL complete culture medium in a cell culture incubator. Eight hours after cell seeding, 500 μL of complete culture medium was gently added into the outer wells to ensure that the culture medium within the assembled matrices was connected with the medium in the outer wells.

2.4. Adhesion assay

After macrophages were allowed to adhere to the matrices for 4 h, the matrices were taken out of the wells, rinsed twice with PBS, and fixed in 4% paraformaldehyde for 30 min at room temperature. When the fixation was complete, the macrophages adhering to the matrices were stained with Hoechst 33342 and analyzed using a TCS SP5 confocal microscope (Leica, Germany). Ten fields from each sample were counted and an average value was reported.

2.5. Morphology observation of macrophages

Macrophages were cultured on the matrices for 24 h and fixed with 2.5% glutaraldehyde at 4°C overnight. After a rinse in deionized water, dehydration in a series of ethanol (30%, 50%, 70%, 80%, 90%, and 100%, 10 min for each step), and a vacuum dry at 37°C, the construct was coated with gold and observed under the SEM. For cell counting, ten fields for each sample were analyzed and the average value was reported.

2.6. Real-time PCR

Total RNA was extracted from the macrophages after they were cultured for 24 h on the smooth and nanofibrous gelatin matrices with an RNeasy Mini Kit, and then reverse transcribed into cDNA using the iScript™ gDNA Clear cDNA Synthesis Kit. The resulting cDNA was prepared for a real-time PCR together with the SYBR® Green Supermix and the gene specific primers: nitric oxide synthase 2 (Nos2) (forward, 5’-CAAGCTGAACTTGAGCGAGGA-3’; reverse, 5’-TTTACTCAGTGCCAG AAGCTGGA-3’), mannose receptor C type 1 (Mrc1) (forward, 5’-AGCTTCATCTTCGGGCCTTTG-3’; reverse, 5’-GGTGACCACTCCTGCTGCTTTAG-3’), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (forward, 5’-CCAATGTGTCCGTCGTGGATCT-3’; reverse, 5’-GTTGAAGTCGCAGGA GACAACC-3’). A CFX96™ real-time system (BioRad, USA) was used to perform the reactions and analyze the results. The relative mRNA expression of target genes was normalized by Gapdh.

2.7. Immunofluorescence staining

After cultured on the smooth and nanofibrous matrices for 24 h, macrophages were fixed in 4% paraformaldehyde for 30 min at room temperature and rinsed in PBS. The macrophages were incubated in 0.3% TritonX-100 (diluted in PBS) for 10 min to permeabilize cell membranes, and blocked in 5% goat serum (diluted in PBS) at room temperature for 2 h. Next the specimens were incubated at 4°C overnight with either an anti-iNOS antibody (1:200 diluted in 5% goat serum) or an anti-Mannose Receptor antibody (1:1000 diluted in 5% goat serum), followed by an Alexa Fluor Plus 555 secondary antibody (1:200 diluted in 5% goat serum) at room temperature for 2 h. The macrophage cytoskeletons were stained with CF633 phalloidin (10 U/mL, diluted in 5% goat serum) for 30 min and the nuclei were labelled with Hoechst 33342 (1 μg/mL, diluted in PBS) for 15 min. After mounted in ProLong™ Gold Antifade Mountant, the constructs were observed under the confocal microscope.

2.8. Multiplex cytokine assay

The culture supernatants were collected after the macrophages were cultured on the matrices for 24 h. The levels of interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), macrophage inflammatory protein 2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) in the supernatants were determined using the Multiplexing LASER Bead Assay (Eve Technologies, Canada).

2.9. Co-culture assay

BMMSCs were isolated from the bone marrow of rats’ femurs as previously described [33]. The BMMSCs at passages 3–5 were seeded in 12-well culture plates at a density of 1 × 105 cells per well in 2 mL complete culture medium and allowed to adhere overnight in a cell culture incubator. The assembled matrices fabricated as described in Section 2.2 and inserted in the 12-well and 1 × 105 macrophages in 500 μL complete culture medium were seeded onto the matrices. Eight hours after macrophage seeding, 500 μL of a complete culture medium was gently added into each well, allowing for free exchange of soluble factors between BMMSCs under the gelatin matrix and the macrophages on the gelatin matrix. After co-cultured with BMMSCs for 24 h, the macrophages on the matrix were harvested for RNA extraction or subjected to immunofluoresence staining as described in Section 2.3 and 2.4.

2.10. RNA-seq and bioinformatic analysis

Total RNA was isolated from macrophages cultured for 24 h on the smooth and nanofibrous matrices using the RNeasy Mini Kit. RNA integrity number (RIN) values greater than 9.0 were used for library preparation and sequencing. The sequencing was performed on the NextSeq 550 platform (Illumina, USA) after the cDNA library was prepared according to the instructions of the Illumina TruSeq® Stranded mRNA LT kit. The initial sequencing data were processed by the Department of Bioinformatics at UT Southwestern Medical Center. The edgeR package from R/Bioconductor was applied over the read counts to evaluate differential expression [34]. Using the smooth gelatin matrix group as a control, the data were analyzed on WebGestalt for Gene Ontology (GO) enrichment and Gene Set Enrichment Analysis (GSEA) [35]. The GO enrichment results were visualized using the GOplot package [36]. Z score for each gene set is calculated as follows: z score = (number of up-regulated genes – number of down-regulated genes) / the square root of the number of all differentially expressed genes in the gene set.

2.12. Statistical analysis

For the analysis of cell adhesion and morphology, ten fields were randomly selected from each sample and a total of 30 sample points in each group were used. For all quantitative analyses, the results were presented as mean ± standard error (n=3). An unpaired Student’s t-test was performed to determine the statistical difference using GraphPad Prism 5.01 (GraphPad Software, USA), and p < 0.05 was considered as significantly different. The edgeR analysis to filter out the differentially expressed genes was performed using R Studio software (available for free at https://www.rstudio.com/).

3. Results

3.1. Macrophages adhered to a greater extent but spread less on the surface of nanofibrous architecture

Gelatin matrices with two distinct topographical features were constructed to examine the effects of nanofibrous architecture on macrophages (Figure 1A). The smooth gelatin matrix had a smooth surface and was used as a control, while the nanofibrous gelatin matrix displayed a network of interwoven nanofibers with an average size of approximately 280 nm, the same range of natural collagen fibers in the body (Figure 1A, B). To precisely control cell culture conditions, the nanofibrous and smooth gelatin matrices were mounted onto CellCrown devices for in vitro experiments (Figure 1C).

Figure 1.

Figure 1.

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Characterization of gelatin matrices with two distinct surfaces. (A) SEM images of smooth and nanofibrous gelatin matrices. (B) The distribution of the diameters of the nanofibrous gelatin matrix. (C) Photo images showing the smooth (left) and nanofibrous (right) gelatin matrices mounted onto CellCrown devices.

As shown in Figure 2 A&B, the number of macrophages adhering on the nanofibrous matrix (240±25 cells/mm2) was twice higher than that on the smooth matrix (634±40 cells/mm2) 4 h after the macrophages were seeded on the matrices. After cultured for 24 h, the number of the macrophages on both matrices increased. However, the number of macrophages on the nanofibrous matrix (1240±98 cells/mm2) remained significantly higher than that on the smooth matrix (760±112 cells/mm2) (Figure 2C, D).

Figure 2.

Figure 2.

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Adhesion and spreading of macrophages on gelatin matrices. (A) Representative images of macrophages on gelatin matrices at 4 h after seeding (Blue: nuclei; Green: gelatin matrices). (B) Quantification of macrophages adhered on gelatin matrices at 4 h after seeding. (C) SEM images of macrophages cultured on gelatin matrices for 24 h. (D) Counts of macrophages on gelatin matrices at 24h after seeding. (E) Quantification of round shaped cell proportions at 24 h after seeding. (n = 3; *p < 0.05, **p < 0.01)

Approximately 80% of the macrophages maintained round shapes on the nanofibrous matrix, while that number was approximately 50% on the smooth matrix (Figure 2C, E). Additionally, some macrophages on the smooth gelatin matrix spread out to form a flat shape. Meanwhile, the average size of the macrophages on the smooth surface was larger than that on the nanofibrous surface (Figure 2C).

3.2. Nanofibrous architecture attenuated the expressions of both M1 and M2 polarization markers

The relative mRNA expression of the M1 polarization marker nitric oxide synthase 2 (Nos2) on the nanofibrous matrix was one tenth as many as that on the smooth matrix (Figure 3A). Also, the immunofluorescence staining of iNOS protein (the Nos2 gene product) in macrophages on the nanofibrous matrix was much weaker than that on the smooth matrix (Figure 3C). On the other hand, the expressions of the M2 polarization marker Mrc1 and the corresponding protein MMR on the nanofibrous matrix were down-regulated compared to those on the smooth matrix (Figure 3B, D).

Figure 3.

Figure 3.

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The polarization of macrophages on gelatin matrices. (A, B) A real-time PCR analysis of macrophage polarization markers on smooth and nanofibrous surfaces. (C, D) The immunofluorescence staining of macrophage polarization markers on smooth and nanofibrous surfaces. (n = 3; *p < 0.05, **p < 0.01)

3.3. Nanofibrous architecture did not affect the inflammatory secretion of macrophages

The concentrations of the inflammation related cytokines in the culture medium did not show significant difference between the nanofibrous and smooth matrices after culturing the macrophages for 24 h (Figure 4). Although there was no statistical significance, the macrophages on the nanofibrous surface showed a trend of higher chemokine (C-C motif) ligand 5 (CCL5) secretion than the macrophages on the smooth surface among all the cytokines tested. Furthermore, secreting levels of macrophage inflammatory protein 2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and granulocyte colony stimulating factor (G-CSF) were all over 5000 pg/mL, while the levels of interleukin-1beta (IL-1β), interleukin-10 (IL-10), macrophage colony stimulating factor (M-CSF) and interferon gamma (IFN-γ) were under 100 pg/mL.

Figure 4.

Figure 4.

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The concentrations of inflammation relevant cytokines after the macrophages were cultured on smooth and nanofibrous gelatin matrices for 24 h. (n = 3)

3.4. Nanofibrous architecture attenuated M1 polarization marker expression of macrophages when co-culturing with MSCs

When exposed to the paracrine influence of MSCs in a co-culture model (Figure 5A), macrophages on the nanofibrous gelatin matrix still displayed lower mRNA expression of Nos2 than on the smooth matrix (Figure 5B), and this tendency was confirmed with the result of iNOS protein staining (Figure 5D). However, the mRNA expression of Mrc1 (Figure 5C) and the staining of its corresponding protein MMR (Figure 5E) were similar on the smooth and nanofibrous surfaces.

Figure 5.

Figure 5.

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The polarization of macrophages on gelatin matrices when co-cultured with MSCs. (A) Illustration of the co-culture model. (B, C) A real-time PCR analysis of macrophage polarization markers. (D, E) Immunofluorescence staining of macrophage polarization markers on the smooth and nanofibrous surfaces. (n = 3; *p < 0.05

3.5. Nanofibrous architecture up-regulated pathways related to ECM-receptor interaction and down-regulated pathways related to pro-inflammation

After the edgeR analysis of the RNA-seq results, a total of 114 differentially expressed genes were identified between the nanofibrous and smooth groups. Among them, 42 genes were up-regulated and 72 genes were down-regulated in the nanofibrous group when compared with the smooth group. Moreover, a GO enrichment analysis showed the differentially expressed genes as enriched into gene sets related to cell-ECM interaction and host defense (Figure 6). In these gene sets, those related to cell-ECM interaction such as “positive regulation of cell adhesion” in the biological process category, “cell adhesion molecule binding” and “extracellular matrix binding” in the molecular function category, z scores were mostly positive. This result indicated that these processes or functions were more likely to be increased in the macrophages on the nanofibrous matrix. However, other gene sets related to host defense, exemplified by the “regulation of inflammatory response”, “positive regulation of defense response”, and “response to molecule of bacterial origin” in the biological process category had mostly negative z scores, indicating that these processes were more likely to be decreased in macrophages on the nanofibrous matrix (Figure 6 and Supplementary Table 1).

Figure 6.

Figure 6.

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A gene ontology enrichment analysis of differentially expressed macrophage genes on the nanofibrous gelatin matrix, using macrophages on the smooth matrix as a control.

The KEGG pathways enriched from the GSEA analysis of the whole transcriptome displayed similar tendency as was shown in the GO enrichment (Figure 7). The ECM-receptor interaction pathway and focal adhesion pathway were up-regulated, and the NF-kappa B and NOD-like receptor signaling pathways along with several other pathways about microorganism infection were all down-regulated on the nanofibrous matrix.

Figure 7.

Figure 7.

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KEGG pathway enrichment results from the GSEA analysis (NES symbolizes a normalized enrichment score and FDR is the false discovery rate)

4. Discussion

Due to the critical role of macrophages in determining the fate of implanted biomaterials, attempt to modulate macrophage behaviors through the intrinsic physiochemical properties of biomaterials has been active in the field of biomaterial research [37–40]. Previous studies using synthetic polymers indicated that the nano-structured topography of biomaterials possessed the potential to regulate macrophage responses [14, 41, 42]. However, an in-depth understanding of how nanofibrous architecture modulates macrophage polarization and the genes related to ECM-receptor interaction and inflammation signaling is absent. In the present study, we evaluated macrophages’ responses to the nanofibrous architecture of a natural ECM derivative by comparing RAW264.7 (a widely used murine macrophage cell line) cultured on nanofibrous and smooth matrices from several aspects including cell morphology, adhesion, polarization, and transcriptome expression. Our results show that the nanofibrous structure down-regulated inflammatory responses of macrophages. The expression of iNOS (Nos2) in macrophages cultured on the nanofibrous gelatin matrix was significantly lower than that on the smooth gelatin matrix, even under the paracrine influence of MSCs (Figure 3A, C; Figure 5B, D). MSCs play a critical role in tissue regeneration, not only for their potential to differentiate into tissue cells but also for their paracrine mechanism to modulate immune cells [43]. Given the importance of MSC-macrophage crosstalk in tissue healing and regeneration [44], we introduced the MSC-macrophage co-culture model to simulate the regenerative microenvironment in vivo, which helps evaluate the effect of the nanofibrous architecture on macrophages from a more biomimetic perspective. It is interesting to note that, although the presence of MSCs tended to mitigate the polarization discrepancy of macrophages caused by different matrix cues (Figure 5), the expression of Nos2 in macrophages cultured on nanofibrous matrix was still significantly lower than that in macrophages on smooth matrix. As a primary marker for M1 polarization, iNOS (inducible nitric oxide synthase) is an important enzyme in charge of producing nitric oxide (NO), which acts as an effector in inflammation in defense of microbial infection [45]. Besides the microbial killing function, NO as an important gaseous transmitter plays a complicated role in inflammation. For example, low amount of NO produced by eNOS (endothelial nitric oxide synthase) alleviated inflammation by preventing the inflammatory cells from adhering and migrating, while relatively high amounts of NO generated by iNOS promoted inflammation by facilitating leukocyte infiltration [46]. A comprehensive analysis of the whole transcriptome in this work further indicated that several pivotal pro-inflammatory pathways (e.g. NF-kappa B signaling and NOD-like receptor signaling) and pathways associated with various types of infectious microbes were down-regulated when the macrophages were cultured on the nanofibrous surface (Figure 7). This result was consistent with the GO enrichment of the differentially expressed genes of macrophages on nanofibrous and smooth surfaces (Figure 6). By matching the differentially expressed genes (Supplementary Table 1) to the marker panels of gene expression associated with macrophage polarization [47], it was shown that M1 polarization associated genes, including TNF alpha induced protein 3 (Tnfaip3), Interleukin 6 (Il6), Nos2, Chemokine (C-X-C motif) ligand 2 (Cxcl2), Macrophage receptor with collagenous structure (Marco), Interleukin 1 beta (Il1b), Interleukin 1 alpha (Il1a), and Interleukin 12B (Il12b), were all down-regulated on the nanofibrous surface. The potential of nanofibrous architecture to inhibit inflammatory reaction of macrophages was also found in synthetic polymers such as poly (L-lactic acid) (PLLA) and polycaprolactone (PCL) [41, 42], suggesting that this effect is independent of substrates.

Interestingly, while macrophages cultured on the nanofibrous surface displayed significantly lower M1 polarization, they did not show a definite tendency toward M2 polarization. In fact, the expression of MMR (Mrc1), a classic M2 marker, in macrophages on the nanofibrous surface was significantly lower than that on the smooth surface (Figure 3B, D). Furthermore, the transcriptome analysis indicated that none of the M2 polarization associated genes (except for Ccl22) was up-regulated on the nanofibrous surface (Supplementary Table 1). These results suggest that the nanofibrous architecture has the tendency to retain macrophages to their original uncommitted M0 state. This effect may be attributed to the reduction of cell contractility when the cells were cultured on a substrate that mimics the architecture of native ECM [48].

Macrophages displayed a more active interaction with the nanofibrous matrix. A higher number of macrophages adhered to the nanofibrous matrix than on the smooth matrix (Figure 2A-D). Accordingly, the transcriptome analysis demonstrated an upward trend in adhesion-related gene sets (e.g. positive regulation of cell adhesion) and pathways (e.g. ECM-receptor interaction and focal adhesion) (Figures 6&7).

Surprisingly, the expressions of membrane receptor genes responsible for collagen adhesion, such as integrin subunit beta 1 (Itgb1), integrin subunit beta 2 (Itgb2), and macrophage scavenger receptor 1 (Msr1) were not up-regulated. Instead, the expressions of integrin subunit alpha V (Itgav), integrin subunit alpha 6 (Itga6), integrin subunit beta 5 (Itgb5), and Cd36 were up-regulated (Supplementary Figure 1) [37]. Additional research is needed to clarify the relationship between the nanofibrous structure and these up-regulated membrane receptors as well as their downstream effects on adhered macrophages.

Based on the GO enrichment analysis, several gene sets associated with cytokines/chemokines (e.g. cytokine secretion, the cytokine-mediated signaling pathway, cytokine receptor activity, leukocyte migration, and cell chemotaxis) appeared when comparing the macrophages on the nanofibrous surface to those on the smooth surface. However, the levels of the representative cytokines/chemokines were not significantly different in the culture supernatants of the macrophages cultured on these two surfaces (Figure 4). This discrepancy suggests that a longer time (>24 h) may be needed for the macrophages to produce and release cytokines and chemokines. RAW264.7 is a cell line with a fast proliferation rate, and a long culturing time results in cell density stress that greatly affects cell state [49]. Meanwhile, a low RAW264.7 seeding density approach to preventing cell overpopulation leads to the formation of patches and clusters, which is not a stable cell state as well. In consideration of the above reasons, the 24-h culture protocol and a median seeding density were adopted in this study, and the results revealed the interaction of macrophages with the nanofibrous architecture at an early stage. In our following work, non-proliferating human blood derived primary macrophages will be used to observe the effect of nanofibrous structure on macrophages for a prolonged period of time. Additionally, an in-vivo evaluation will give us an in-depth understanding of the interaction between ECM-mimicking matrices and macrophages.

5. Conclusions

Nanofibrous architecture promotes macrophage adhesion, and the adhered macrophages on the nanofibrous surface tended to retain an original non-polarized state. Moreover, nanofibrous architecture has the potential to minimize inflammatory responses. A whole transcriptome analysis revealed that the nanofibrous architecture up-regulated the pathways related to ECM-receptor interaction and down-regulated pathways related to pro-inflammation. The inflammation-inhibiting and adhesion-promoting effects of nanofibrous architecture can be utilized for the design and development of novel immunomodulatory biomaterials for tissue regeneration.

Supplementary Material

1

Acknowledgments

Funding

This study was partially supported by NIH/NIDCR (R01DE024979 to X. Liu) and by National Natural Science Foundation of China (No.81530050 to F.-M. Chen) and China Scholarship Council (No. 201703170163 to R.-X. Wu).

Footnotes

Conflicts of interests

The authors declare no conflicts of interest.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information files or are available from the authors upon request.

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