Shear and Dynamic Compression Modulates the Inflammatory Phenotype of Human Monocytes in vitro

Monocytes and their derived macrophages are found at the site of remodeling tissue, such as fracture hematoma, that is exposed to mechanical forces and have been previously implicated in the reparative response. However, the mechanoresponsive of monocytes and macrophages to skeletal tissue-associated mechanical forces and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of skeletal tissue-associated loading conditions to modulate human monocyte activation and phenotype. Primary human monocytes or the human monocyte reporter cell line, THP1-Blue, were encapsulated in agarose and exposed to a combination of shear and compressive loading for 1 h a day for 3 consecutive days. Exposure of monocytes to mechanical loading conditions increased their pro-inflammatory gene and protein expression. Exposure of undifferentiated monocytes to mechanical loading conditions significantly upregulated gene expression levels of interleukin(IL)-6 and IL-8 compared to free swelling controls. Additionally, multiaxial loading of unstimulated monocytes resulted in increased protein secretion of TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) compared monocytes cultured under free-swelling conditions. This modulatory effect was observed irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli lipopolysaccharide and interferon-γ or the M2/anti-inflammatory differentiation factor interleukin-4. Furthermore, mechanical shear and compression were found to differentially regulate nitric oxide synthase 2 (NOS2) and IL-12B gene expression as well as inflammatory protein production by THP1-Blue monocytes. The findings of this study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. This may play a role in healing pathways that are mechanically regulated. An in depth understanding of the impact of skeletal tissue-associated mechanical loading on monocyte behavior may identify novel targets to maximize inflammation-mediated repair mechanisms.

Monocytes and their derived macrophages are found at the site of remodeling tissue, such as fracture hematoma, that is exposed to mechanical forces and have been previously implicated in the reparative response. However, the mechanoresponsive of monocytes and macrophages to skeletal tissue-associated mechanical forces and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of skeletal tissue-associated loading conditions to modulate human monocyte activation and phenotype. Primary human monocytes or the human monocyte reporter cell line, THP1-Blue, were encapsulated in agarose and exposed to a combination of shear and compressive loading for 1 h a day for 3 consecutive days. Exposure of monocytes to mechanical loading conditions increased their pro-inflammatory gene and protein expression. Exposure of undifferentiated monocytes to mechanical loading conditions significantly upregulated gene expression levels of interleukin(IL)-6 and IL-8 compared to free swelling controls. Additionally, multiaxial loading of unstimulated monocytes resulted in increased protein secretion of TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) compared monocytes cultured under free-swelling conditions. This modulatory effect was observed irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli lipopolysaccharide and interferon-γ or the M2/anti-inflammatory differentiation factor interleukin-4. Furthermore, mechanical shear and compression were found to differentially regulate nitric oxide synthase 2 (NOS2) and IL-12B gene expression as well as inflammatory protein production by THP1-Blue monocytes. The findings of this study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. This may play a role in healing pathways that are mechanically regulated. An in depth understanding of the impact of skeletal tissue-associated mechanical loading on monocyte behavior may identify novel targets to maximize inflammation-mediated repair mechanisms.

INTRODUCTION
The repair process following traumatic injury to the musculoskeletal system is known to be influenced by the mechanical environment. The natural course of bone healing can be intramembranous ossification resulting from stable fracture fixation and subsequent low interfragmentary motion, or endochondral ossification which is associated with moderate interfragmentary movement (1). In addition to driving fracture healing responses, mechanical forces of an appropriate magnitude are also key to maintaining cartilage homeostasis within the articulating joint (2).
A wound healing response is initiated during the process of bone fracture repair, as well as marrow stimulation techniques applied in cartilage repair strategies where the subchondral bone is penetrated. This involves inflammatory cell exudation or infiltration to the site of injury, followed by coagulation activation and fibrin clot formation, which is known to regulate monocyte chemotaxis and proliferation (3,4). Monocytes and monocyte-derived macrophages are key immune effector cells playing a vital role in host defense, as well as contributing to tissue remodeling and repair (5). Macrophages are associated with a high degree of plasticity having the potential to change phenotype in response to environmental cues, and may be classified according to pro-inflammatory (M1) or anti-inflammatory (M2) subsets (5). Pro-inflammatory macrophages are associated with high production of pro-inflammatory cytokines and increased microbicidal activity, whereas anti-inflammatory macrophages are associated with wound healing and immunoregulatory functions (3,6).
Infiltrating monocytes and macrophages may influence the success of musculoskeletal tissue repair processes. Macrophage depletion has been previously demonstrated to negatively impact endochondral ossification and subsequently delay bone fracture healing (7)(8)(9). Furthermore, monocyte and macrophage associated inflammatory cytokines such as IL-6 and TNFα are known to promote bone repair, with inhibition of TNF-α signaling shown to delay both intramembranous and endochondral bone formation (10,11). In contrast to bone healing, pro-inflammatory factors such as IL-1β and TNFα produced by both activated monocytes and M1 polarized macrophages, induce destructive processes in cartilage tissue including catabolic enzyme expression and reduced extracellular matrix deposition (12)(13)(14). However, recruitment of antiinflammatory macrophages to the site of subchondral drill holes within osteochondral defects using chitosan-glycerol phosphate composites was reported enhance subchondral bone repair and improve cartilage resurfacing, further highlighting the impact of macrophages on skeletal tissue repair (15). Monocytes and macrophages are found at the site of remodeling tissue that is exposed to mechanical forces and have been previously implicated in the reparative response (16-18). As the area of osteoimmunology gains in importance, the influence of mechanical stimulation on immune cell phenotype needs to be investigated in greater detail. However, the responsiveness of macrophages and monocytes, their lineage precursors, to mechanical forces that are native to skeletal tissues and the effect of such mechanical stimuli on macrophage phenotype requires further elucidation. Therefore, the aim of this study was to investigate the impact of mechanical shear and compressive loading on monocyte activation and phenotype. Unstimulated, M1 or M2-stimulated primary human monocytes as well as the human monocyte reporter cell line, THP1-Blue, were exposed to a combination of shear and compressive loading in vitro. Gene expression levels of inflammatory mediators and inflammatory protein secretion was assessed following 3 days of mechanical stimulation.

Human Monocyte Isolation
Human monocytes were isolated from buffy coats left over from voluntary whole blood donations after informed consent of the donors according to the regulations of Swiss Red Cross Blood Service. Buffy coats were processed within 23 h after blood donation by centrifugation at 5,000 g for 15 min and subsequent separation on Compomat G5 (Fresenius, Oberdorf, Switzerland) using top-and-bottom 450 ml blood bag systems pre-filled with Citrate-Dextrose-Phosphate Solution (Fresenius). Buffy coats were anonymized prior to delivery from the Blood Service to AO Institute in line with the ethics code provided by the Swiss Drug Law (Heilmittelgesetz). For the isolation of peripheral blood mononuclear cells (PBMCs), each buffy coat was diluted at a 1:5 ratio with 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Thirty milliliter of diluted buffy coat was layered on 15 ml of Ficoll and centrifuged at 1,000 g for 15 min without brake. Following centrifugation, the interphase layer containing PBMCs was removed and washed with 0.5% BSA/PBS containing 2 mM EDTA. Isolated PBMCs were labeled with 100 µl of anti-CD14 magnetic bead solution (Miltenyi Biotec Bergisch Gladbach, Germany) in the dark at 4 • C for 20 min.
Monocytes were isolated utilizing MACS LS Separation columns and a MidiMACS TM Separator (Miltenyi Biotec), according to manufacturer's instructions. Purity of isolated CD14+ cells was assessed by fluorescence activated cell sorting (FACS) analysis. 1 × 10 5 monocytes were incubated with APC-Cy7-conjugated anti-human CD14 antibody (BD pharmingen) for 20 min in the dark at 4 • C. FACS analysis was performed using a BD Aria III machine, and data analyzed using BD FACS Diva 6.1.3 software (BD Biosciences). The average purity of CD14+ monocytes from all donors was found to be 95% (data not shown). Monocytes were isolated from two individual buffy coat donors and pooled per experiment.

Agarose Gel Seeding and Culture
To evaluate the effect of mechanical loading on monocyte phenotype, human primary monocytes and THP1-Blue cells were encapsulated in 2% low melting temperature agarose (Lonza) at a cell density of 3 × 10 6 monocytes per gel. In brief, a 4% agarose solution was prepared by dissolving low melting temperature agarose in sterile phosphate buffered saline and heated. The 4% agarose solution was cooled and mixed with an equal volume of cells suspended in pre-warmed culture media, composed of RPMI-1640 medium supplemented with 2 mM L-glutamine, 1% penicillin/streptomycin and 10% heat inactivated FBS. Two hundred and fifty microliters of cells/agarose suspension was added to a sterile cap of an Eppendorf tube and gels were allow to set at 37 • C for 20 min. All agarose constructs were carefully removed from the Eppendorf cap, placed in a sterile PEEK sample holder and cultured with 2.5 ml of culture medium. To investigate the effect of mechanical loading on macrophage phenotype, CD14+ monocytes were stimulated with 10 ng/ml IFN-γ (PeproTech, Rocky Hill, NJ, USA) and 100 ng/ml lipopolysaccharide to induce differentiation toward a pro-inflammatory/M1 phenotype, 10 ng/ml IL-4 (PeproTech) for an anti-inflammatory/M2 phenotype or unstimulated for 72 h prior to loading. Agarose gels containing THP1-Blue monocytes were prepared 24 h prior to loading.

Mechanical Loading
CD14+ monocyte seeded agarose gels were mechanically loaded using a custom built multi-axial load bioreactor based on a 32 mm ceramic hip ball that can apply compression, shear or a combination of the two, to the sample as previously described (19,20). Shear (±25 • ball rotation at 1 Hz) and compression (10% compression superimposed on top of a 10% pre-strain at 1 Hz) loading was applied for 1 h a day for 3 consecutive days. This protocol was chosen as it has been shown to direct osteochondral differentiation of human MSCs and therefore we aimed to investigate similar loading patterns on the modulation of macrophage phenotype (21). Control gels were maintained in free-swelling conditions for the duration of the experiment. To investigate the effect of shear or compression alone on monocyte phenotype, THP1-Blue monocytes were stimulated with shear or compression alone as well as multiaxial loading for 1 h a day for 3 consecutive days. Control gels were maintained in free-swelling conditions for the duration of the experiment. Cell culture media was refreshed every 24 h prior to loading.

Secreted Alkaline Phosphatase Assay
Secreted embryonic alkaline phosphatase (SEAP) levels were detected in cell culture supernatant using a QUANTI-Blue TM enzymatic assay (InvivoGen) according to manufacturer's instructions. SEAP levels were determined qualitatively following spectrophotometric measurement at 620 nm.

Statistical Analysis
IBM SPSS Statistics 21.0 (IBM, New York, USA) and GraphPad Prism software version 6 (GraphPad Software Inc., La Jolla, USA) were used for all statistical analysis. To take donor variability into account between primary monocyte donors, mixed models analysis was applied to test for statistical differences between loaded and free-swelling groups with monocyte donor considered a random factor. THP1-Blue monocyte data sets were analyzed using a Kruskal-Wallis test followed by Dunn's multiple comparisons test. For all analyses, differences were considered statistically significant at P < 0.05.

Pro-inflammatory Gene and Protein Expression by Differentially Activated Primary Human Monocytes Following Multiaxial Loading
Monocytes encapsulated in 2% agarose gel were unstimulated, LPS and IFN-γ or IL-4-stimulated for 3 days, prior to subjection Human interleukin-8 (IL8) C-X-C motif chemokine ligand 8 (CXCL8) Hs00174103_m1 Human interleukin-10 (IL10) Cytokine Synthesis Inhibitory Factor (CSIF) Hs00961622_m1 Human interleukin-6 (IL6) B-Cell Stimulatory Factor (BSF)-2 Hs00985639_m1 Human tumor necrosis factor (TNF) Tumor Necrosis Factor (TNF)-α Hs01113624_g1 Human  Figure 1A). Additionally, IL-4 stimulated monocytes were associated with significantly higher CCL18 expression compared to LPS and IFN-γ stimulated monocytes (41.5-fold increase), confirming their polarization toward an M2-like phenotype. Unstimulated primary human monocytes significantly upregulated gene expression levels of the pro-inflammatory genes IL6 (5.9-fold change) and IL8 (2.8-fold change) following 3 days of mechanical loading compared to monocytes cultured in free-swelling conditions ( Figure 1B). Additionally, expression of the antiinflammatory macrophage marker IL10 was decreased in all four donors compared to free-swell controls, with gene expression levels undetectable in donors 1 and 3 following loading. No significant difference was observed in the expression levels of inflammatory mediators CCL18, TNF, and CCL2. Although a similar trend was observed toward inflammatory gene expression by LPS and IFN-γ activated monocytes following mechanical loading, larger variation was observed between donors and these findings were not statistically significant ( Figure 1C). However, expression of CCL2 was significantly decreased (1.9-fold decrease). Additionally, gene expression levels of IL10 were also undetectable in LPS and IFN-γ stimulated monocytes from donors 1 and 3 following loading. In a similar manner to unstimulated monocytes, IL-4 activated cells were also associated with a significant increase in IL6 (8.9-fold change) and decrease of IL10 (3.1-fold) expression ( Figure 1D). Compared to free-swelling controls, mechanical loading of unstimulated monocytes significantly increased production of the pro-inflammatory mediators TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) (Figure 2). Protein levels of IL-10, CCL18, IP-10, MCP-1, MDC, and IL-1β produced by loaded unstimulated monocytes did not significantly differ from free-swelling controls. In a similar manner to gene expression levels, a trend toward an increase in IL-6 production was observed in response to loading of unstimulated monocytes. However, large donor variation was observed and this finding was not statically significant. Mechanical stimulation of LPS and IFN-γ stimulated monocytes significantly increased MDC levels in addition to TNF-α, MIP-1α, and IL-13 (Figure 2). In a similar manner to gene expression data, IL-4 activated monocytes were associated with significantly increased production of proinflammatory factors IL-6, IL-8, TNF-α, MIP-1α, IP-10, IL-13, IL-1β as well as IL-10 in response to mechanical loading, and decreased expression of CCL18 and MDC (Figure 2).

Inflammatory Gene and Protein Expression by THP1-Blue Monocytes Following Mechanical Shear or Compression
To evaluate the potential of mechanical shear or compression to differentially regulate inflammatory gene and protein expression by human monocytes, unstimulated THP1-Blue monocytes were subjected to multiaxial loading conditions, or mechanical shear or compression alone. THP1-Blue monocytes significantly upregulated gene expression levels of the pro-inflammatory markers NOS2 and IL12B in response to compression alone compared to the combination of compression and shear, as well as shear alone following 3 days of loading (Figure 3). Gene expression levels of IL6, IL-8, TNF-α, PTGS2, IL-10, CCL2, and CCL18 did not significantly differ between loading conditions, or compared to free-swelling controls at this time point. However, significantly increased levels of TNF-α, IL-10, IL-8, IL-13, and MDC were detected in the cell culture media harvested from gels subjected to the combination of compression and shear, as well as compression and shear alone for 3 days compared to control ( Figure 4A). Additionally, the application of compressive loading alone significantly upregulated IL-1β production by monocytes, whereas shear alone increased the release of MCP-1 compared to all other culture conditions. Levels of IL-6 and IP-10 did not significantly differ in response to any loading condition compared to free-swelling cultures. In addition to modulating inflammatory cytokine production, the application of both compression and shear or shear alone induced the release of secreted alkaline phosphatase by THP1-Blue monocytes, indicative of NF-κB and AP-1 transcription factor activation ( Figure 4B).

DISCUSSION
Monocytes and their derived macrophages are considered key players in tissue remodeling and repair processes. Mechanical loading has been previously shown to influence the levels or pro and anti-inflammatory macrophages in a model of tendon healing (18). Additionally, cyclic strain has been reported to modulate macrophage polarization state toward a reparative phenotype which promoted extracellular matrix synthesis (22). Monocytes are found at the site of skeletal tissue injuries resulting from either traumatic bone fractures, or microdrilling of the subchondral bone to facilitate microfracturemediated cartilage repair (23). However, the mechanoresponsive of monocytes and their derived macrophages to skeletal tissue-associated mechanical forces, and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of shear and compressive forces to modulate human monocyte activation and phenotype. In the present study, exposure of monocytes to mechanical loading conditions increased their production of pro-inflammatory mediators. Furthermore, mechanical loading modulated the production of inflammatory factors produced by monocytes irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli LPS and IFN-γ or the M2/anti-inflammatory differentiation factor IL-4.
Bone fractures associated with less mechanical stability are known to heal via the process of endochondral ossification, involving inflammation, callus formation and tissue remodeling processes (1). Infiltration of macrophages into the fracture callus occurs at an early stage of fracture healing, and inhibition of macrophage recruitment impairs vascularization, decreases callus formation and delays repair (24). Macrophages are associated with a high degree of plasticity and can change phenotype according to environmental cues, encompassing both pro-inflammatory/M1 and anti-inflammatory/M2 phenotypes (5). In an experimental osteotomy model, M1-polarized macrophages were identified as the primary macrophage phenotype in the osteotomy area 24 h post-surgery (7). Interestingly, M1 polarized macrophages have also been reported to promote the osteogenic differentiation of bone  marrow-derived mesenchymal stem cells (MSCs) (25). Furthermore, macrophage infiltration and prevalence of a M1-like phenotype has been observed in association with MSC-mediated bone repair in vivo (26,27). In the present study, we have observed a skewing of monocyte activation toward a M1-like phenotype following 3 days of shear and compressive loading, highlighting the responsiveness of human monocytes to mechanical stimuli. These findings may shed some light on how the biomechanical environment may play a role in guiding monocyte/macrophage polarization, and potentially contribute to skeletal tissue repair.
In the present study, we have observed an increase in TNFα, MIP-1α, and IL-13 protein production by unstimulated as well as LPS and IFN-γ and IL-4 activated monocytes following mechanical shear and compression. Two of four donors in the unstimulated group also substantially increased IL-6 production upon loading. Furthermore, levels of the pro-inflammatory cytokines IL-1β, IL-8, and IL-6 produced by IL-4 activated monocytes were increased following loading. Additionally, gene expression levels of IL-8 and IL-6 were increased by unstimulated primary human monocytes subjected to the combination of mechanical compression and shear. This could indicate that these factors would be induced within an unstable fracture. Previous reports have highlighted an influence of mechanical stimuli resulting from fracture fixation stability upon gene expression of matrix metalloproteinase (MMP)-9 and MMP-13 by fracture hematoma in rats (28). Both MMP-9 and MMP-13 are known to play a key role during the process of endochondral bone formation, facilitating extracellular matrix and cell migration (29). Additional studies have demonstrated an upregulation in the expression of genes involved in cartilage and skeletal development by callus tissue following mechanical stimulation, in a rat osteotomy model (30). However, the impact of mechanical stimuli upon the induction of inflammatory gene expression by fracture hematoma in vivo requires further investigation. Production of IL-6 and TNF-α is characteristic of activated monocytes and pro-inflammatory M1 macrophages (5,31). Both IL-6 and TNF-α signaling are known to play a key role in bone fracture healing (32,33). Additionally, TNFα is involved in osteoclastic bone resorption (34). Interestingly, in addition to acting as a chemotactic cytokine for monocytes and neutrophils, IL-8 has been reported by Ringe et al. to induce migration of human MSCs (35,36). Furthermore, IL-8 is known to promote angiogenesis (37,38). Mechanical stimulation of early human fracture hematoma has also been previously reported to result in increased production of the pro-angiogenic protein vascular endothelial growth factor (VEGF) (39). MIP-1α, also known as CCL3, has been previously implicated in the recruitment of macrophages to the site of injury during bone repair (40). In contrast to the observed upregulation of proinflammatory mediators by monocytes in response to mechanical loading, we also detected increased levels of IL-13 protein irrespective of cell activation with LPS and IFN-γ or IL-4. The pleiotropic cytokine IL-13 is known to polarize macrophages toward an M2 phenotype, encompassing anti-inflammatory and tissue repair subsets (41). Additionally, IL-13 is a key mediator of tissue fibrosis, reported to stimulate transforming growth factor (TGF)-β1 production by monocytes and macrophages, as well as increasing TGF-β1 activation (42,43). TGF-β signaling may promote extracellular matrix deposition and tissue remodeling (44,45). In addition to mediating tissue fibrosis and macrophage polarization, a role for IL-13 in osteoclast differentiation and bone resorption has been previously highlighted (46). Macrophages have been previously reported to change their phenotype throughout the course of bone healing, with a more predominant role of the M2 subset identified at later stages of repair (7). Given that in the current study loading of monocytes also resulted in production of the M2-polarization factor IL-13, whether a longer duration of loading may switch the balance from M1/M2 requires further investigation.
Having identified an influence of mechanical loading upon the phenotype of M1 or M2-differentiated as well as undifferentiated primary human monocytes, we next sought to investigate whether shear forces or compression alone may be responsible for this effect. In addition to compressive loading, cartilage in the articulating joint and fractures that have not been rigidly fixated, are also subjected to shear. Therefore, we next sought to examine whether shear or compression alone may exert differential effects on undifferentiated monocytes, to gain further insight into whether loading associated with various skeletal tissues may differentially modulate monocyte activation. The human monocyte cell line THP1-Blue TM was utilized to assess the effect of shear, compression or the combination of both on inflammatory mediator expression by monocytes. THP1-Blue TM cells are a reporter cell line, which express secreted embryonic alkaline phosphatase (SEAP) following activation of the transcription factors NF-κB and AP-1. Both NF-κB and AP-1 are activated in monocytes following toll-like receptor 4 (TLR4) stimulation and are involved in the induction of inflammatory gene expression (47). In a similar manner to primary human monocytes, the application of both compression and shear increased expression of inflammatory mediators TNFα, IL-13, macrophage-derived chemokine (MDC), and IL-10. Interestingly, IL-10 is an anti-inflammatory cytokine but is also produced by monocytes in response to pro-inflammatory stimulation a part of a regulatory feedback mechanism (48). Furthermore, IL-10 is a factor also known to induce the differentiation of macrophages toward an anti-inflammatory phenotype (5). MDC is chemotactic for monocytes and is also considered a marker of M2 macrophages (49,50). Additionally, we observed differential effects of compression or shear alone on monocyte phenotype. Application of compression alone increased expression of the pro-inflammatory genes IL12B and NOS2 compared to shear alone or the combination of both stimuli. Interestingly, inducible nitric oxide synthase, which is encoded by the gene NOS2, has been previously shown to be expressed the initial phase of bone fracture repair (51). Compression alone also significantly increased IL-1β production compared to control, whereas shear alone was found to increase MCP-1. Furthermore, stimulation with both compression and shear or shear alone significantly increased SEAP expression compared to free-swelling controls, suggestive of potential TLR4 activation by monocytes in response to shear force (47). TLR4 has been previously implicated in the pro-inflammatory response of chondrocytes to high fluid shear, and increasing evidence highlights a role of TLR4 activation in inflammatory and catabolic processes associated with osteoarthritis pathogenesis (52)(53)(54). Our present findings may provide further insight to the mechanism of the effect of mechanical loading on monocyte activation, however further investigation is required to evaluate the effect of such mechanical stimuli directly on monocyte TLR expression and activation. These mechanically induced changes suggest that the initial monocyte containing hematoma would respond to mechanical motion by upregulating proinflammatory cytokines. This could be a danger signal that recruits cells to the site of damage and regulates their response. Rigid fixation would reduce this inflammatory signal leading to a different response. However, further investigation is required to fully determine the impact of mechanical stimuli resulting from fracture fixation stability upon monocyte behavior in vivo, and the subsequent influence of mechanically-stimulated monocytes upon skeletal tissue repair.
The findings of the present study highlight the mechanical sensitivity of human monocytes to skeletal tissue-associated loading conditions. Monocyte-derived macrophages have been previously shown to respond to mechanical strain in vitro, with an observed upregulation of MMP-1 and MMP-3 expression as well as the transcription factors c-fos and c-jun (55). Furthermore, Yang et al. highlighted a potential role of mechanical strain in the induction of monocyte to macrophage differentiation, mediated by upregulation of the monocyte differentiation-associated transcription factor PU.1 (55). In line with our findings, shear stress has also been shown to promote macrophage differentiation toward a pro-inflammatory M1-like phenotype in a model of atherosclerosis (56). Interestingly, extracellular physical cues resulting from surface stiffness have been reported to modulate TLR signaling by macrophages (57). However, whether these signaling pathways play a role in the responsiveness of monocytes and monocyte-derived macrophages to shear and compressive forces native to skeletal tissue requires further elucidation. This study has several limitations. Primary peripheral blood monocytes treated with LPS and IFN-γ or IL-4 were used as a model in vitro culture system to evaluate the effect of loading on M1 or M2-polarized cells, respectively. Investigation of the effect of mechanical loading on M1 and M2 pre-differentiated macrophages and a longer duration of study may be required to specifically examine the modulatory effect of mechanical loading on macrophage polarization state. Additionally, 2% agarose was used in this study as a cell-carrier system in our in vitro model to investigate the short-term response of human monocytes to skeletal tissue-associated mechanical stimuli. However, previous studies have highlighted an impact of different scaffold materials toward the cellular mechanical response (58). Therefore, further investigation may be required to determine whether monocyte interactions with different scaffold materials such as fibrin gels, as a more specific model of the wound healing phase of tissue repair, may determine their response to such mechanical stimuli. Furthermore, additional examination utilizing in vivo models of fracture healing is required to relate this observed induction of inflammatory mediators by mechanical loaded monocytes to skeletal tissue repair.
In conclusion, the findings of the present study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. An in depth understanding of the impact of skeletal tissueassociated mechanical loading on monocyte behavior and their subsequent influence on local cellular responses and tissue repair processes, may identify novel strategies to maximize inflammation-mediated repair mechanisms. Furthermore, the findings of this study may provide insights for the development of novel rehabilitation medicine strategies to improve therapeutic outcome for skeletal tissue repair.

DATA AVAILABILITY
The datasets generated for this study are available on request to the corresponding author.

AUTHOR CONTRIBUTIONS
NF performed the experiments, processed samples, analyzed, and interpreted the data. UM processed samples and analyzed the data. MA supported in data analysis and interpretation. MS conceived and designed the study, and interpreted the data. NF, UM, MA, and MS drafted and critically revised the manuscript for important intellectual content. All authors have approved the final submitted manuscript.

FUNDING
This work was supported by the AO Foundation and the Swiss National Science Foundation (Grant no. 31003a_146375/1).