High-Purity Magnesium Staples Suppress Inflammatory Response in

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High-purity Mg Staples Suppress Inflammatory Response in Rectal Anastomoses Jiazeng Xia, Hui Chen, Jun Yan, Hongliu Wu, Hao Wang, Jian Guo, Xiaonong Zhang, Shaoxiang Zhang, Changli Zhao, and Yigang Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00813 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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ACS Applied Materials & Interfaces

High-purity Mg Staples Suppress Inflammatory Response in Rectal Anastomoses

Jiazeng Xia,1 Hui Chen,2 Jun Yan,3 Hongliu Wu,4 Hao Wang,1 Jian Guo,1 Xiaonong Zhang,4 Shaoxiang Zhang,5 Changli Zhao,*,4,† and Yigang Chen*,1,† 1

Department of General Surgery, Wuxi Second Hospital, Nanjing Medical University,

Jiangsu 214002, PR China 2

Department of Pathology, Nanjing General Hospital, Jiangsu 210002, PR China

3

Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth

People’s Hospital, Shanghai 200233, PR China 4

State Key Laboratory of Metal Matrix Composites, School of Materials Science and

Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China 5

Suzhou Origin Medical Technology Co. Ltd., 2 Haicheng Road, Changshu Economic

and Technology Development Zone, Jiangsu 215513, PR China

†

Both authors contributed equally to this work.

Corresponding authors *Yigang Chen, Department of General Surgery, Nanjing Medical University Affiliated Wuxi Second Hospital, 68 Zhongshan Road, Wuxi 214002, PR China. Tel./fax: +86-510-66681222. E-mail address: [email protected]. *Changli Zhao, State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, No. 800 Dongchuan Road, Shanghai 200240, PR China. Tel./fax: +86 213420 2759. E-mail address: [email protected]. KEYWORDS: biodegradable materials, magnesium, anastomoses, staples, inflammation

1

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ABSTRACT: Magnesium-based materials are promising biodegradable implants, although the impact of magnesium on rectal anastomotic inflammation is poorly understood. Thus, we investigated the inflammatory effects of high-purity Mg staples in rectal anastomoses by in vivo luciferase reporter gene expression in transgenic mice, hematoxylin-eosin staining, immunohistochemistry, and western blotting. As expected, strong IL-1β-mediated inflammation and inflammatory cell infiltration were observed 1 day after rectal anastomoses were stapled with high-purity Mg or Ti. However, inflammation and inflammatory cell infiltration decreased more robustly 4-7 days post-operation in tissues stapled with high-purity Mg. This rapid reduction in inflammation was confirmed by immunohistochemical analysis of IL-6 and TNF-α. Western blot also suggested that the reduced inflammatory response is due to suppressed TLR4/NF-κB signaling. In contrast, MCP-1, uPAR, and VEGF were abundantly expressed, in line with the notion that expression of these proteins is regulated by feedback between the VEGF and NF-κB pathways. In vitro, expression of MCP-1, uPAR, and VEGF was also similarly high in primary rectal mucosal epithelial cells exposed to extracts from Mg staples, as measured by antibody array. Collectively, the results suggest that high-purity Mg staples suppress the inflammatory response during rectal anastomoses via TLR4/NF-κB and VEGF signaling.

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INTRODUCTION

Since the 1960s, an increasing number of anastomosis instruments have been used in rectal reconstruction surgery.1 These staples generally simplify and increase the efficiency of the necessary surgical procedures. However, complications remain an ongoing issue,2-3 foreign-body reactions being one of the risk factors.4 Currently, nonabsorbable pure titanium or titanium alloy staples are the most widely used. Although these staples are strongly biocompatible, long-term retention in the body may elicit local inflammation and other complications.5-6 For instance, congestion and inflammation are often observed by postoperative colonoscopy in rectal anastomoses closed with Ti staples. Owing to excellent biocompatibility and biodegradability, magnesium and magnesium alloys are also regarded as promising implants.7-9 In particular, magnesium-based materials have been extensively characterized as bone implants and vascular stents with favorable mechanical properties.8-10 Use of magnesium alloys in the gastrointestinal tract has also recently attracted attention. For example, Anja et al.11 reported that the biodegradable Mg alloy WZ21 is suitable as a component (tip) of short-term rivets used in gastrointestinal interventions. Chu et al.12 found that coating magnesium alloy wires with organic gelatin–hydroxyapatite improves corrosion resistance to simulated intestinal fluid. Of note, magnesium ions are essential for all life as cofactor for ATP and polyphosphates such as DNA and RNA, and as secondary messenger in intracellular signaling.13 Mg2+ ions also suppress production of inflammatory cytokines.14-15 However, the relationship between magnesium and inflammation in rectal anastomoses has not been investigated extensively. In this study, high-purity magnesium staples were used to anastomose rectal tissue in IL-1β luciferase reporter mice, and the ensuing inflammatory response was evaluated in the surrounding tissue. The inflammatory effects of extracts from these staples were also assessed in vitro in primary rectal mucosal epithelial cells. Ti staples were used as control. The data demonstrate that magnesium staples inhibit inflammation in rectal anastomosis. 3



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EXPERIMENTAL SECTION Materials. High-purity Mg (99.98 wt.%) staples were supplied by Suzhou

Origin Medical Technology Co. Ltd., China (Figure 1). The yield tensile strength of these staples was 147 ± 8 MPa, with ultimate tensile strength 196 ± 5 MPa and elongation 14.6 ± 5 %.16 Medical Ti staples (Ti–3Al–2.5V Alloy, Ethicon Endo-Surgery, Inc.) were used as control. The weight, thickness, and surface area were 0.9 mg, 0.26 mm, and 9.5 mm2, respectively, for one high-purity Mg staple, whereas the weight and surface area were 2.5 mg and 8.66 mm2 for one medical Ti-6Al-4V staple. All samples were packed and sterilized with 29 kGy 60Co radiation. Extracts were prepared according to ISO 10993-5. Briefly, Mg and Ti staples were immersed for 24 h at 37 ºC in high-glucose Dulbecco’s Modified Eagle’s medium (GibcoTM, USA) supplemented with 10 % fetal bovine serum (Hyclone, USA), and in a humidified atmosphere with 5 % CO2. The ratio of the staple surface area to the extraction medium was 1.25 cm2/mL. Extracts were removed and centrifuged, and the resulting supernatant was collected and stored at 4 ºC. In vivo inflammation. Rectal anastomosis in IL-1β luciferase reporter mice. Animal experiments were consistent with the Guidance Suggestions for the Care and Use of Laboratory Animals issued by Ministry of Science and Technology, People’s Republic of China, and were approved by the Ethics Committee of Nanjing Medical University Affiliated Wuxi Second Hospital. IL-1β luciferase reporter mice express firefly luciferase via a 4.5-kb fragment of the human IL-1 promoter.17 These mice are used to study local acute inflammation, as IL-1β is one of the important mediators.18-20 Thus, 30 male IL-1β luciferase reporter mice, age 7 weeks and weighing 22 ± 2.5 g, were obtained from Shanghai Research Centre for Model Organisms. These mice were randomly assigned to receive 1 mm incisions in the rectum that were then closed with either Mg or Ti staples (Figure 1). Surgery was performed under anesthesia, which consisted of an intraperitoneal injection of sodium pentobarbital (0.1 mg/g body weight). In vivo luciferase was imaged daily up to 7 d post-surgery using an IVIS imaging system (QuickView3000, Bio-Real, Austria). At 1, 4, and 7 d, five mice were euthanized, and tissues 4


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surrounding implants were collected, stained with hematoxylin-eosin, and analyzed by immunohistochemistry and western blot. Hematoxylin-eosin staining and immunohistochemistry. Specimens were fixed in 10 % buffered formaldehyde, sectioned at 4 µm, mounted on glass slides, and stained with hematoxylin-eosin. For immunohistochemistry, sections were deparaffinized in xylene, and rehydrated in graded concentrations of ethyl alcohol (100, 95, and 75 %) and then in water. Specimens were then microwaved twice at 99 ºC for 6 min in citrate buffer pH 6.0, immersed in 3 % H2O2 for 10 min to inhibit endogenous peroxidase, washed three times with phosphate-buffered saline for 5 min each, and blocked for 10 min at room temperature with normal mouse serum. Sections were then probed for 24 h at 4 ºC with antibodies against IL-6 (DR6139, 1:100, UCallM, China), TNF-α (RPN075, 1:100, UCallM, China), MCP-1 (MR0076, 1:400, UCallM, China), GRO (AF5403, 1:500, Affinity, USA), and uPAR (10286-1-AP, 1:500, Proteintech, USA). Subsequently, sections were washed three times with phosphate-buffered saline for 10 min each, labeled with biotinylated anti-mouse or anti-rabbit immunoglobulin, stained with 3,3-diaminobenzidine tetrahydrochloride, and imaged on a light microscope with MICRO IMAGETM software (Olympus Optical Corp. Ltd., Japan). Expression was quantified in Motic Fluo 1.0 software (Motic China Group Co. Ltd., China). Western blot. Total protein was extracted using Qproteome Mammalian Protein Prep Kit (37901, Qiagen, Germany), and quantified by Bradford assay (P0006, Beyotime, China) according to the manufacturer’s protocol. Typically, 30 µg protein was resolved by SDS-PAGE, and transferred onto nitrocellulose membranes. Membranes were then blocked for 1 h at room temperature with 5 % non-fat dry milk, and probed at 4 ºC overnight with rabbit antibodies against human TLR4 (AF7017, 1:1000, Affinity, USA), phosphorylated NF-κB p65 (RLP0191, 1;500, Ruiying, China), and VEGF (DR7128, 1:500, UCallM, China). To control for loading, membranes were also probed with an antibody against β-actin (60008-1-Ig, 1:1000, Proteintech, USA). Finally, membranes were visualized with an enhanced chemiluminescence system (Beyotime, China) according to the manufacturer’s 5



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instructions, and analyzed in QuantityOne software (BioRad, USA). Inflammation in vitro. Primary cell culture. Experiments were compliant with Chinese laws and guidelines on informed consent. Collection and use of human specimens were approved by the Institutional Review Board at Nanjing Medical University Affiliated Wuxi Second Hospital. Patients, most of whom were convalescing from respiratory illnesses in an ambulatory hospital, were selected and biopsied. Specimens were minced, centrifuged at 800 rpm for 5 min, and the resulting supernatant was discarded. The sediment was incubated for 30 min at 37 ºC in an incubator with humidified 5 % CO2 air, centrifuged at 800 rpm for 5 min, and resuspended. Dissociated cells were then harvested by sequential filtration. To remove fibroblasts, filtered cells were incubated on glass Petri dishes three times for a total of 45 min. Non-adherent cells were counted, and diluted to a density between 5 × 105 and 1 × 106/mL. When examined under an inverted microscope, the cells were found to be morphologically intact, clearly bounded, homogeneous, and transparent. The cells also expressed cytokeratin 18, as detected by immunofluorescence (BM1770, Wuhan Boster Biological Technology, Ltd., China), and were polygonal or fusiform with typical cobblestone-like appearance (Figure 2). Third-generation cells were used in experiments. Antibody array. Antibody arrays were used to assess the effects of staple extracts in vitro. Briefly, primary rectal mucosal epithelial cells were seeded in 96-well plates at 2 × 104 cells/well, and incubated with 100 µL extracts from Mg and Ti staples. After 1 day, cells were lysed, and screened with RayBio® Human Angiogenesis Antibody Array G-Series 1000 (RayBiotech Inc., USA, http://www.raybiotech.com) to semi-quantitatively measure levels of 43 different inflammation and angiogenesis factors, including ANG (angiogenin), EGF (epidermal growth factor), ENA-78 (epithelial neutrophil-activating peptide-78), bFGF (basic fibroblast growth factor), GRO (growth-related oncogene), IFN-γ (interferon-gamma), IGF-1 (insulin-like growth factor-1), IL-6 (interleukin-6), IL-8 (interleukin-8), leptin, MCP-1 (monocyte chemotactic

protein-1),

PDGF-BB

(platelet-derived

growth

factor

B-chain

homodimer), PLGF (placenta growth factor), RANTES (normal T-cell expressed and 6


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secreted), TGF-β1 (transforming growth factor beta-1), TIMP-1 (tissue inhibitor of metalloproteinase-1), TIMP-2 (tissue inhibitor of metalloproteinase-2), THPO (thrombopoietin), VEGF-A (vascular endothelial growth factor-A ), VEGF-D (vascular endothelial growth factor-D ), ANGPT-1 (angiopoietin-1), ANGPT-2 (angiopoietin-2), ANGSTN (angiostatin), COL18A1 (collagen type XVIII, alpha 1), G-CSF (granulocyte colony-stimulating factor), GM-CSF (granulocyte macrophage colony-stimulating

factor),

I-309

(T

lymphocyte-secreted

protein),

IL-10

(interleukin-10), IL-1α (interleukin-1 alpha), IL-1β (interleukin-1 beta), IL-2 (interleukin-2), IL-4 (interleukin-4), I-TAC (interferon-inducible T cell alpha chemoattractant), MCP-3 (monocyte chemotactic protein-3), MCP-4 (monocyte chemotactic protein-4), MMP-1 (matrix metalloproteinase-1), MMP-9 (matrix metalloproteinase-9), PECAM-1 (platelet/endothelial cell adhesion molecule-1), Tie-2 (transmembrane tyrosine kinase, receptor of angiopoietin), TNF-α (TNF alpha), uPAR (urokinase receptor), VEGF R2 (vascular endothelial growth factor receptor 2), and VEGF R3 (vascular endothelial growth factor receptor 3). Western blot. Antibody array results were verified by western blot. Briefly, primary rectal mucosal epithelial cells were extracted with Qproteome Mammalian Protein Prep Kit (37901, Qiagen, Germany), and total protein was measured by Bradford assay (P0006, Beyotime, China) following the manufacturer’s protocol. Extracts were analyzed by standard western blotting using appropriate antibodies, visualized with an enhanced chemiluminescence system (Beyotime, China) according to the manufacturer’s instructions, and analyzed in QuantityOne software (BioRad, USA). Statistical analysis. Luciferase activity (photons/s) at stapled sites was quantified using LivingImage software (Xenogen, USA) and ImagePro Plus 7 (Media Cybernetics, USA). Data are reported as mean ± standard deviation, and were analyzed in SPSS 18.0 (SPSS Inc., USA) by paired-samples t test. One-way analysis of variance was performed to test differences among groups at each time point, although nonparametric K independent samples Kruskal-Wallis test was performed when equal variance could not be assumed. p < 0.05 was considered statistically 7



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significant.

RESULTS Inflammation in vivo. IL-1β luciferase expression. IL-1β luciferase reporter

mice are used as dynamic models to observe acute inflammation.17 Thus, we monitored luciferase expression daily up to 7 d in mice anastomosed with Mg or Ti staples (Figure 3A). Expression was similar between groups 1 and 2 d post-surgery, and clearly decreased from 4 to 7 d as inflammation subsided. However, IL-1β luciferase activity during this period was significantly lower in tissues surrounding high-purity Mg staples than in tissues surrounding Ti staples (Figure 3B). Hematoxylin-eosin staining. There were no unexpected deaths following surgery, and rectal anastomotic fistula was not observed. Representative rectal tissues surrounding Mg and Ti staples at 1, 4, and 7 d post-operation are shown stained with hematoxylin-eosin in Figure 4. Severe inflammation was noted 1 d after implantation of Mg and Ti staples, but this reaction had subsided at 4 and 7 d. As can be seen in the figure, a smaller area was infiltrated with fewer inflammatory cells in tissues surrounding Mg staples than in tissues surrounding Ti staples, indicating that the former suppressed inflammation. Immunohistochemistry. Expression of IL-6 and TNF-α, as measured by immunohistochemistry, was similar 1 d post-surgery in tissues surrounding Mg and Ti staples (Figure 5). Expression generally decreased in both groups over time, but was significantly lower in tissues stapled with high-purity Mg. In contrast, expression of MCP-1 was significantly higher in tissues surrounding Mg staples at 1, 4, and 7 d (Figure 6A), as can be seen in representative immunohistochemical images (Figure 6). Similarly, uPAR expression was significantly higher at 1 and 4 d (Figure 6B), although GRO expression was significantly lower at 1 d (Figure 6C). However, GRO expression was comparable between groups 4 and 7 d post-operation. These results were confirmed by integrated optical density (Figure 6D). Western blot for TLR4, NF-κB p-p65, and VEGF. Expression of TLR4, phosphorylated NF-κB p65, and VEGF in rectal tissues surrounding Mg and Ti 8


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staples at 1, 4, and 7 d post-operation is shown in Figure 7. VEGF was expressed 3.92-, 3.61-, and 4-fold higher 1, 4, and 7 days after surgery, respectively, in tissues stapled with Mg than in tissues surrounding Ti staples. On the other hand, expression of TLR4 and phosphorylated NF-κB p65 was comparable between groups at 1 d, but was 3.5 and 2.4 times lower at 4 and 7 d in the former. Based on the data, Mg staples appear to activate VEGF signaling more robustly than they suppress NF-κB signaling. In vitro inflammation. Antibody array. RayBio® chips containing multiple antibodies against inflammatory cytokines and chemokines were used to characterize cells exposed to extracts from Mg and Ti staples (Figure 8). The inflammatory chemokines MCP-1 (9.6-fold) and uPAR (2.5-fold) were more strongly expressed in the former, along with the angiogenesis factor VEGF-A (5.9-fold, p < 0.05, Figure 8B). In contrast, expression of the inflammatory chemokine GRO was significantly lower (0.3-fold, p < 0.05). There were no statistical differences in expression of all other inflammatory cytokines and chemotaxis, angiogenesis, and growth factors. Western blot. To confirm results from antibody arrays, expression of select proteins was assessed by western blotting, using antibodies against MCP-1 (MR0076, 1:500, UCallM, China), VEGF-A (DR7128, 1:1000, UCallM, China), GRO (AF5403, 1:1000, Affinity, USA), and uPAR (10286-1-AP, 1:1000, Proteintech, USA). As shown in Figure 9, cells exposed for 24 h to extracts from Mg staples expressed MCP-1, VEGF, and uPAR more abundantly. In contrast, these cells expressed GRO at significantly lower levels, confirming results from RayBio arrays.

DISCUSSION

Inflammation is triggered when the body detects foreign substances, a response that is in part the result of both the local tissue injury and the immunological reaction to it.21-24 Indeed, strong IL-1β-mediated inflammation and infiltration by inflammatory cells were observed in tissues stapled with Mg or Ti 1 d after rectal anastomosis surgery in mice. Accordingly, the inflammatory factors IL-6 and TNF-α, which are key triggers of the inflammatory response,25-26 were abundantly expressed. We note that IL-6 is produced by several types of immune cells, including mesothelial cells 9



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and lymphocytes, usually in response to stimuli such as TNF-α, IL-1β, and bacterial endotoxins.27 On the other hand, TNF-α is produced mainly by macrophages, lymphoid cells, and neuronal cells, typically in response to lipopolysaccharide and other bacterial products.28-29 Thus, expression of IL-6 and TNF-α correlates with the severity of inflammation, and are prognostic of many inflammatory diseases.30-31 Owing to the protective role of the immune system under normal conditions, postoperative inflammation is tightly regulated and generally subsides with time,32-33 as we observed 4-7 d after surgery. Notably, the inflammation subsided more rapidly in tissues stapled with Mg than in tissues stapled with Ti. These results suggest that post-operative inflammation is suppressed more effectively in tissues surrounding Mg staples. The main degradation products of high-purity Mg staples are Mg2+ ions, hydroxyl, and hydrogen. Of these, Mg is an essential nutrient that also inhibits inflammation.14-15 Accordingly, the biological activity of magnesium has attracted more attention. For example, Mg-based materials were found to increase expression of calcitonin gene-related polypeptide-α, transcription factors, and integrin subunits, to alter the cell cycle, and to induce apoptosis via the mitogen-activated protein kinase pathway.34-36 In addition, increased extracellular Mg reduces the inflammatory reaction in rats, whereas Mg deficiency elicits inflammatory syndrome, activates leukocytes and macrophages, and releases inflammatory cytokines.37-39 In vivo measurements indicated that Mg staples effectively downregulated TLR4 and NF-κB, in line with our previous results suggesting that extracts of Mg alloy inhibit NF-κB signaling in intestinal epithelial cells.16 TLR4 is a “molecular sentry” that rapidly activates NF-κB signaling in response to tissue damage.40-42 As a result, activation or suppression of TLR4/NF-κB signaling also activates or suppresses the inflammatory response. Accordingly, inhibition of TLR4/NF-κB signaling by high-purity Mg staples also resulted in decreased inflammation and expression of the downstream genes IL-6 and TNF-α, in line with previous reports demonstrating that Mg inhibits inflammation via NF-κB.14-15 For instance, Zhai et al.43 reported that metallic magnesium can suppress NF-κB signaling pathway during osteoclast-induced osteolysis, and Witte et 10


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al.44 demonstrated that Mg-based scaffolds modulate the host inflammatory response when implanted in the rabbit distal femur condyle. Hydrogen, another major degradation product, is also known to be an important physiological regulator with anti-inflammatory and anti-apoptotic effects in cells and organs.45-46 For instance, hydrogen was shown to regulate the inflammatory mediators IL-1β, IL-6, and TNF-α in the injured small intestine.47-48 Strikingly, the protective effects of hydrogen are also associated with decreased NF-κB signaling.49-50 Therefore, we hypothesize that hydrogen and Mg ions released from Mg staples synergize to suppress TLR4/NF-κB signaling, diminish IL-6 and TNF-α expression, and, ultimately, inhibit local inflammation. Notably, Mg staples also induced VEGF expression both in vitro and in vivo. This result is significant, as the vascular reaction is central to inflammation,51-52 and as VEGF is an important mediator of inflammation in the brain, lung, and rectum.53-55 Indeed, abundant expression of VEGF promotes angiogenesis and microvascular permeability, which, in turn, facilitate the actions of local inflammatory products.56-57 In addition, VEGF is not only a downstream target of TLR4/NF-κB signaling, but is also a feedback inhibitor.58 In particular, VEGF suppresses the TLR4/NF-κB cascade in macrophages via phosphatidylinositide-3 kinase/protein kinase B and suppressor of cytokine signaling-1,59-60 and thereby alleviates inflammation. Notably, high levels of VEGF also induced abundant expression of the inflammatory chemotaxis factors MCP-1 and uPAR, as measured in vitro by antibody array and in vivo by immunohistochemistry. This phenomenon was previously observed in endothelial cells,61 and is also associated with local inflammation.62-63 Indeed, MCP-1 is one of the most important chemotaxis factors that regulate migration and infiltration of monocytes, memory T lymphocytes, and natural killer cells.64 Chemotaxis factors also mediate leukocyte adhesion to the vascular wall, as well as leukocyte migration.65 Hence, it is possible that by upregulating MCP-1 and uPAR, high-purity Mg staples both recruit anti-inflammatory cells such as monocytes and protect against bacterial infection. We note that Mg staples likely exert both suppressive and activating effects on 11



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MCP-1 and uPAR expression. On one hand, the staples should suppress expression by inhibiting NF-κB signaling,66 but should also induce expression by stimulating VEGF expression.67-68 However, it appears that the stimulatory effects of VEGF are more dominant, as antibody arrays, in vivo immunohistochemistry, and in vitro western blot all suggest that the staples boost expression of both chemotactic factors. In summary, high-purity Mg staples appear to elicit less severe TLR4/NF-κB signaling and local inflammation. In particular, degradation of the staples into Mg2+ ions and H2 appears to suppress TLR4/NF-κB signaling, thereby decreasing expression of the inflammatory factors IL-6 and TNF-α, and relieving the inflammatory response. In addition, Mg staples boost expression of VEGF, which has direct and indirect anti-inflammatory effects. High levels of the chemotaxis factors MCP-1 and uPAR due to VEGF activation further reinforce the anti-inflammatory effects. The proposed mechanism by which Mg staples regulate the inflammatory response in rectal anastomosis is illustrated in Figure 10.

CONCLUSIONS

High-purity Mg was investigated in vitro and in vivo as a biodegradable anastomotic staple that may potentially regulate inflammation. The data indicate that high-purity Mg staples suppress the inflammatory response by regulating TLR4/NF-κB signaling and VEGF expression.

AUTHOR INFORMATION

Corresponding Authors *Email: [email protected] (Y. Chen) and [email protected] (C. Zhao). ORCID：：0000-0002-3036-3537 Notes The authors declare that they have no conflict of interest.

ACKNOWLEDGMENTS

This work was supported by Jiangsu Provincial Commission of Health and Family Planning Research Project (No. H201545), by Wuxi Hospital Administration Center 12


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Q.

Comparison

of

the

Effects

of

Mg-6Zn

and

Ti-3Al-2.5V

Alloys

on

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Figure captions Figure 1. High-purity Mg and Ti staples. (A) Anastomotic staples made of high-purity Mg and Ti. (B) A Mg staple used to close a rectal incision in an IL-1β luciferase reporter mouse. Figure 2. Primary rectal mucosal epithelial cells immunostained for cytokeratin 18. Figure 3. In vivo IL-1β luciferase reporter activity 1 to 7 d post-operation in mice anastomosed with Mg and Ti staples. (A) Colors indicate photons/sec/cm2/steradian according to the color scale. (B) Whole-body luciferase activity (photons × 106/sec). Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. Figure 4. Representative rectal tissues surrounding Mg and Ti staples, collected 1, 4, and 7 d post-operation, and stained with hematoxylin-eosin (100×). Inset images show inflammatory cells (400×) (A) Number of infiltrating inflammatory cells at 1, 4, and 7 d. (B) Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. Figure 5. Expression of inflammatory factors IL-6 and TNF-α 1, 4, and 7 d post-operation, as measured by immunohistochemistry. (A and B) Representative tissues (200×). (C) Quantification. Data are mean ± SD, and were analyzed using Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. Figure 6. Expression of MCP-1, uPAR, and GRO at 1, 4, and 7 d post-operation, as measured by immunohistochemistry. (A-C) Representative tissues (200×). (BD) Quantification. Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. Figure 7. Expression of TLR4, NF-κB, and VEGF in rectal tissues surrounding Mg and Ti staples 1, 4, and 7 d post-operation. (A) Representative western blots. (B) Quantification. Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. Figure 8. RayBio Human Angiogenesis Antibody Arrays were used to investigate expression of inflammatory and angiogenesis factors in cells exposed to extracts of Mg and Ti staples. (A) Expression of 43 inflammatory and angiogenesis factors. (B) 18


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Higher levels of MCP-1, VEGF-A, and uPAR, but lower levels of GRO, were observed in cells exposed for 24 h to extracts from Mg staples. Figure 9. Protein levels of MCP-1, VEGF, uPAR, and GRO, as assessed by western blot. Consistent with results from antibody arrays, MCP-1, VEGF, and uPAR were more abundantly expressed in cells exposed to extracts from Mg staples, but GRO expression was diminished. (A) Representative western blots. (B) Quantification. Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. Figure 10. Proposed mechanism by which high-purity Mg staples regulate inflammation through VEGF and TLR4/NF-κB. The staples release Mg2+ ions and H2 to induce high expression of VEGF, as indicated by red arrows. At the same time, TLR4/NF-κB signaling is suppressed, as indicated by blue lines. High levels of VEGF have direct (blue lines) and indirect anti-inflammatory effects through a feedback loop between VEGF and TLR4/NF-κB (green lines). As a result, levels of the inflammatory factors IL-6 and TNF-α diminish. In addition, high levels of the inflammatory chemotaxis factors MCP-1 and uPAR, which are suppressed by TLR4/NF-κB but stimulated by VEGF, enhance the anti-inflammatory effects. Ultimately, the staples inhibit inflammation in rectal anastomosis.

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Figure 1. High-purity Mg and Ti staples. (A) Anastomotic staples made of high-purity Mg and Ti. (B) A Mg staple used to close a rectal incision in an IL-1β luciferase reporter mouse. 57x74mm (300 x 300 DPI)



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Figure 2. Primary rectal mucosal epithelial cells immunostained for cytokeratin 18. 701x216mm (72 x 72 DPI)


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Figure 3. In vivo IL-1β luciferase reporter activity 1 to 7 d post-operation in mice anastomosed with Mg and Ti staples. (A) Colors indicate photons/sec/cm2/steradian according to the color scale. (B) Whole-body luciferase activity (photons × 106/sec). Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. 61x80mm (300 x 300 DPI)



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Figure 4. Representative rectal tissues surrounding Mg and Ti staples, collected 1, 4, and 7 d post-operation, and stained with hematoxylin-eosin (100×). Inset images show inflammatory cells (400×) (A) Number of infiltrating inflammatory cells at 1, 4, and 7 d. (B) Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. 108x103mm (300 x 300 DPI)


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Figure 5. Expression of inflammatory factors IL-6 and TNF-α 1, 4, and 7 d post-operation, as measured by immunohistochemistry. (A and B) Representative tissues (200×). (C) Quantification. Data are mean ± SD, and were analyzed using Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. 280x286mm (72 x 72 DPI)



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Figure 6. Expression of MCP-1, uPAR, and GRO at 1, 4, and 7 d post-operation, as measured by immunohistochemistry. (A-C) Representative tissues (200×). (BD) Quantification. Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. 387x276mm (72 x 72 DPI)


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Figure 7. Expression of TLR4, NF-κB, and VEGF in rectal tissues surrounding Mg and Ti staples 1, 4, and 7 d post-operation. (A) Representative western blots. (B) Quantification. Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. 169x154mm (120 x 120 DPI)



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Figure 8. RayBio Human Angiogenesis Antibody Arrays were used to investigate expression of inflammatory and angiogenesis factors in cells exposed to extracts of Mg and Ti staples. (A) Expression of 43 inflammatory and angiogenesis factors. (B) Higher levels of MCP-1, VEGF-A, and uPAR, but lower levels of GRO, were observed in cells exposed for 24 h to extracts from Mg staples. 686x708mm (72 x 72 DPI)


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Figure 9. Protein levels of MCP-1, VEGF, uPAR, and GRO, as assessed by western blot. Consistent with results from antibody arrays, MCP-1, VEGF, and uPAR were more abundantly expressed in cells exposed to extracts from Mg staples, but GRO expression was diminished. (A) Representative western blots. (B) Quantification. Data are mean ± SD, and were analyzed by Student’s t test. *, p < 0.05; **, p < 0.01 vs Ti. 170x71mm (300 x 300 DPI)



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Figure 10. Proposed mechanism by which high-purity Mg staples regulate inflammation through VEGF and TLR4/NF-κB. The staples release Mg2+ ions and H2 to induce high expression of VEGF, as indicated by red arrows. At the same time, TLR4/NF-κB signaling is suppressed, as indicated by blue lines. High levels of VEGF have direct (blue lines) and indirect anti-inflammatory effects through a feedback loop between VEGF and TLR4/NF-κB (green lines). As a result, levels of the inflammatory factors IL-6 and TNF-α diminish. In addition, high levels of the inflammatory chemotaxis factors MCP-1 and uPAR, which are suppressed by TLR4/NF-κB but stimulated by VEGF, enhance the anti-inflammatory effects. Ultimately, the staples inhibit inflammation in rectal anastomosis. 318x393mm (72 x 72 DPI)


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High-Purity Magnesium Staples Suppress Inflammatory Response in

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