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- Ben Amara, Heithem, 1984
(författare)
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Biodegradable magnesium implants, immunomodulation, and tissue repair/regeneration
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The century-old paradigm of holding fractures with a metallic implant to enable bone repair, known as osteosynthesis, is still used today without alteration. Patients are increasingly being treated with metallic implants made of magnesium (Mg) that secure osteosynthesis and are reabsorbed in situ without the surgical re-entry that requires their permanent analogs. Often, Mg implants achieve osteosynthesis. However, when failure occurs, aberrant inflammation in overlaying soft tissue and persistent peri-implant radiolucencies generated by gas release from the implants are common. How can this be reconciled with popular concepts predestining Mg implants to promote bone formation by mitigating inflammation and bone resorption? This thesis investigated the sequence of biological processes prompting soft tissue and bone to accommodate Mg implants with different degradation behaviors from early to relatively long healing. Detailed studies of cells and their molecular circuits during inflammation were undertaken in different but related biological compartments surrounding the implants. Complementary analytical microscopy and compositional spectroscopy were performed to characterize tissue assembly at the interface with the implants and beyond. Compared to nondegradable titanium implants, Mg implants amplify initial inflammation in soft tissue and bone. The rapid release of degradation products, including Mg2+ and gas, correlatively induces a strong, transient proinflammatory environment that fosters mRNA and protein programs associated with macrophage polarization, chemotaxis, osteoclastogenesis, and neovascularization but without cytotoxic effects. Thereafter, inflammation markedly subsides. The transition to soft tissue and bone repair coincides with the attenuation of Mg2+ concentrations and gas void generation in the peri-implant milieu in tandem with an enrichment in calcium and phosphorous on the implant surface. Immunomodulation by Mg implants, reflected by a shift from proinflammatory to prohealing macrophage activation, reinforces their anchorage in bone and alleviates fibrotic encapsulation in soft tissue. However, this restorative effect is not equal in response to the various Mg degradation behaviors. Pure Mg implants, which degrade faster than clinical-grade alloyed Mg implants, alter the composition of interfacial bone and result in a previously unknown proadipogenic response in the bone marrow beyond the bone–implant interface. This increased adiposity is closely associated with persistent gas voids in bone marrow. Gas voids encourage inflammation in their microenvironment, trigger mechanosensation, and may induce local bone matrix deposition. In conclusion, Mg implants in different tissues transiently amplify the initial immune reaction, and degradation product escape creates an inflammatory microenvironment at the tissue–implant interface and beyond. An appropriate reparative response is obtained but can be impaired by the uncontrolled implant degradation. Above the demand for rigorous tailoring of Mg implants, healing monitoring needs to expand to tissues outside the confines of the implant interface, with pending questions on the fate of tissues under compromised conditions.
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| 2. |
- Ben Amara, Heithem, 1984, et al.
(författare)
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Bone healing around biodegradable Magnesium implants: Differential response between interfacial and near-implant bone in vivo
- 2022
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Ingår i: 14th Symposium on Biodegradable Metals, Alicante, Spain 24-29 August 2022.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: By virtue of their mechanical properties and of their degradation, magnesium (Mg)-based osteosynthesis systems are metallic implants that hold the fractured bones while eliminated in situ, thus offering the promise of reduced complications posed by permanent implants. A growing amount of research validated Mg-based implants for bone fixation by providing robust evidence in support of new bone deposition in contact with the interfacial degradation layer. Whereas Mg-degradation products are known to distribute in the implant environment, less attention has been paid to the bone response at distance from the implant interface. The present study investigated the structural, cellular, and molecular events taking place at the bone-Mg implant interface and at distance from it after in vivo implantation in an experimental rat model. METHODS: Following approval by the Local Ethical Committee at the University of Gothenburg (Dnr: 14790/2019), male Sprague-Dawley rats (n=56) were implanted with miniature screws manufactured from pure magnesium (99.99% - high purity; Mg) or from pure titanium (grade 4; Ti) (herein, serving as a control, enabling osseointegration in this model). In each animal, the metaphysis of the left and right tibiae was drilled prior to the insertion of Ti or Mg screws. After 3 and 28 days, animals were euthanized, and two types of samples were retrieved (Fig. 1): 1-Implants and peri-implant bone for quantitative polymerase chain reaction (qPCR) (n=8/group/time-point): were separately collected and allocated for molecular gene expression of the implant-adherent cells and of the cells in the peri-implant space. 2-Peri-implant bone with implants en bloc for paraffin or plastic embedding (n=6/group/time-point): enabling radiographical analyses using micro-computed tomography (micro-CT) and histomorphometrical measurements of the bone at the implant interface and at distance from it. Statistical comparisons were made between experimental groups at each time point and between time-points for each experimental group. (Kruskal-Wallis, Mann-Whitney and Wilcoxon signed-rank tests; p<0.05). RESULTS: While histological observations provided evidence of new bone formation at the vicinity of both Ti and Mg, the bone marrow at distance from the implant-interface featured morphological differences between groups (Fig. 2). At 3 days, the proportion of the interstitial and microvascular area was significantly higher at the expense of the area occupied by the hematopoietic cells in Mg- vs Ti-implanted metaphyses. At 28 days, bone marrow around Mg implants showed significantly higher adiposity in comparison to Ti implants. Yet, no differences in the trabecular bone micro-architecture were detected between biomaterials by micro-CT analysis at distance from the implant-interface. The RNA extracted from cells from the implant surface and from the peri-implant bone revealed good quality, allowing detailed molecular analysis. CONCLUSIONS: In comparison to non-degradable Ti controls, the degradation of Mg implants changes the composition of the peri-implant bone marrow, but yet without alteration of new bone formation at the implant interface. ACKNOWLEDGEMENTS: Mg rods were generously provided by the Helmholtz-Zentrum Hereon, Geesthacht, Germany. This project is part of the European Training Network within the framework of Horizon 2020 Marie Skłodowska-Curie Action No 811226.
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| 3. |
- Ben Amara, Heithem, 1984, et al.
(författare)
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Dissecting the sequential interaction between biodegradable magnesium implants and soft tissues in vivo
- 2022
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Ingår i: Materials Science and Engineering Congress, Dredsen, Germany, 27-29 September 2022.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Magnesium-based biomaterials are developed with the intention to enable tissue regeneration while being degraded under physiological conditions and eventually eliminated from the body. Once in contact with tissues, the biodegradability and the biocompatibility of magnesium implants (Mg) are governed by the direct interactions with their immediate milieu. The precise mechanisms through which the soft tissue micro-environment shapes the behaviour of Mg and the host-response remain elusive. Here, it is demonstrated that Mg degradation modulates the initial acute immune response and the subsequent fibrous encapsulation upon subcutaneous implantation in rats monitored at 1-, 3-, 6-, 14- and 28-days following surgery. In comparison to titanium implants (Ti), the initial profuse release of Mg degradation products activates pro-inflammatory pathways through increased recruitment of inflammatory cells to the soft tissue/implant interface and upregulation of pro-inflammatory genes, in parallel with a superior neo-angiogenesis and vascularization at Mg. After 6d, a shift in Mg degradation kinetics dissipates the initial pro-inflammatory response and facilitates the assembly of a comparatively thinner fibrous tissue capsule than around Ti. The reduction in the fibrous encapsulation around the Mg implant aligns with a superior expression of anti-fibrotic marker FOXO-1 at the tissue interface with Mg versus Ti. Mg induce an initial potent yet transient inflammatory response, which is associated with less adverse fibrous encapsulation after tissue healing. Tailoring Mg with controlled initial degradation appears to be crucial to enabling a successful coupling between inflammation and tissue repair during the early host response to Mg.
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| 4. |
- Ben Amara, Heithem, 1984, et al.
(författare)
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Immunomodulation by biodegradable Mg-implants promotes soft and hard tissues responses in vivo
- 2023
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Ingår i: Scandinavian Society of Biomaterials conference, 21–24 March 2023, Røros, Norway.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: Magnesium (Mg)-based degradable implants are an attractive treatment solution for musculoskeletal injuries, avoiding second-stage surgical removal. In multiple clinical applications, the implant is in contact with both the bone and the overlying soft tissue. Although Mg implants are often presented to hold anti-inflammatory properties, less attention has been paid to the sequential response to these implants including initial immune response and subsequent tissue repair. The present study investigated the molecular, cellular, and structural events taking place at the Mg implant interface to soft tissue and bone after in vivo implantation in dedicated experimental rat models. METHODS: Male Sprague Dawley rats received disc-shaped implants in the dorsum subcutis or screw-shaped implants in the proximal tibial metaphysis. Implants were manufactured from pure magnesium (99.99% - high purity; Mg) or from pure titanium (grade 4; Ti) as control. Animals were euthanized after 1, 3, 6, 14, and 28 day of soft tissue implantation, and after 3 and 28 days of bone implantation. Two types of samples were collected: 1-Implants with the adherent cells (n=7-8/group/time-point). These were allocated for cell counting and /or gene expression analyses of implant-adherent cells. 2-Peri-implant tissue with implants (n = 8/group/time-point). These enabled histological and histomorphometric analyses of the fibrous capsule organization around implants inserted in soft tissues and of osseointegration parameters at the bone-implant interface. Statistical comparisons between experimental groups were run using Kruskal-Wallis, and Mann-Whitney tests (p<0.05). RESULTS: Cells adherent to the surface of the implants featured different gene regulation patterns between Mg and Ti groups (Fig. 1). Consistently in soft tissue and in bone, macrophage polarization markers indicated higher expression of proinflammatory macrophage gene inducible nitric oxide synthase (iNos) initially at Mg versus Ti (3 d in bone and 1-6 d in soft tissue). Afterward, gene expression of both macrophage subtypes markers (proinflammatory – iNos and prohealing – Mannose receptor c1; Mrc1) was comparable between implants, irrespective of their insertion site. Histomorphometry evidenced superior bone-implant contact (at 28 d in bone) and thinner fibrous capsule (at 6-28 d in soft tissue) for Mg versus Ti. CONCLUSIONS: In comparison to non-degradable Ti, both soft tissue and bone responses to biodegradable Mg featured an initial yet transient gene activation of the macrophage proinflammatory subtype. Such immunomodulation by Mg resulted in the reduction of fibrous encapsulation in soft tissue and in the promotion of bone formation at the bone-implant interface. ACKNOWLEDGEMENTS: Mg implants were generously provided by Helmholtz-Zentrum Hereon, Geesthacht, Germany. This project is part of the European Training Network within the framework of Horizon 2020 Marie Skłodowska-Curie Action No 811226.
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| 5. |
- Ben Amara, Heithem, 1984, et al.
(författare)
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In vivo interaction between biodegradable magnesium implants and soft tissue Part II: Kinetics of the cellular response at the host-implant interface
- 2021
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Ingår i: 13th Biometal Conference, 23-26 August 2021, Virtual Conference..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: Regenerative therapies often engage multiple tissues. Soft tissue complications (e.g. dehiscences and infection) may violate successful bone regeneration. Magnesium (Mg)-based degradable implants is a promising treatment alternative for musculoskeletal injuries, avoiding second-stage surgical removal. In several clinical applications, the implant is in contact with both the bone and the overlying soft tissue. Whereas the bone response to Mg implants has been a major research focus, less attention has been paid to the soft tissue response. The present study investigated the spatial and temporal molecular, cellular and structural events taking place at the soft tissue-Mg implant interface after in vivo implantation in an experimental rat model. METHODS: Following approval by the Local Ethical Committee at the University of Gothenburg (Dnr 02437/2018), female Sprague-Dawley rats (n=90) were implanted with discs manufactured from pure magnesium (99.99% - high purity; Mg) or from pure titanium (grade 4; Ti) (herein, employed as a control, possessing biocompatibility properties). Subcutaneous pockets were surgically created in the animal dorsum and were implanted with: 1- Ti; or 2- Mg discs; or 3- left without implants (Sham Ti or Sham Mg). After 1, 3, 6, 14 and 28 days, animals were euthanized, and three types of samples were retrieved: 1-Implants with the adherent cells (n=8/group/time-point): for cell counting and molecular gene expression of the implant-adherent cells. 2-Peri-implant exudate (n=8/group/time-point): for analyses of the number, type, viability, and gene expression of cells in the peri-implant space. 3-Peri-implant tissue with implants (n=8/group/time-point): enabling histological and histomorphometric analyses of soft tissue and fibrous capsule organization around the implant. Statistical comparisons were made between experimental groups at each time point and between time-points for each experimental group. (Kruskal-Wallis, Mann-Whitney and Wilcoxon signed-rank tests; p<0.05). RESULTS: Cells recruited to the exudates and adherent to the surface of the implants featured different kinetics between Mg and Ti groups. At the surface of Mg implant, the number of adherent cells sharply increased from 1 day to reach a peak at 6 days, thereafter decreasing toward 28 days. The ratio of implant-adherent/exudate cells was significantly higher at Mg vs Ti after 6 days, whereas the reverse was detected after 28 days. RNA extracted from cells from the different compartments revealed good quality, allowing detailed molecular analysis. After 28d, the fibrous capsule around Mg implants was significantly thinner than around Ti. CONCLUSIONS: In comparison to non-degradable Ti controls, soft tissue healing around biodegradable Mg implants is characterized by an early, intense, but yet transient, cellular influx in the immediate vicinity of the implant surface, and, at later stage, with a reduced fibrotic encapsulation. ACKNOWLEDGEMENTS: Mg implants were generously provided by the Helmholtz-Zentrum Hereon, Geesthacht, Germany. This project is part of the European Training Network within the framework of Horizon 2020 Marie Skodowska-Curie Action No 811226.
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| 6. |
- Ben Amara, Heithem, 1984, et al.
(författare)
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Magnesium implant degradation provides immunomodulatory and proangiogenic effects and attenuates peri-implant fibrosis in soft tissues
- 2023
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Ingår i: Bioactive Materials. - : Elsevier BV. - 2452-199X. ; 26, s. 353-369
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Tidskriftsartikel (refereegranskat)abstract
- Implants made of magnesium (Mg) are increasingly employed in patients to achieve osteosynthesis while degrading in situ. Since Mg implants and Mg2+ have been suggested to possess anti-inflammatory properties, the clinically observed soft tissue inflammation around Mg implants is enigmatic. Here, using a rat soft tissue model and a 1-28 d observation period, we determined the temporo-spatial cell distribution and behavior in relation to sequential changes of pure Mg implant surface properties and Mg2+ release. Compared to nondegradable titanium (Ti) implants, Mg degradation exacerbated initial inflammation. Release of Mg degradation products at the tissue-implant interface, culminating at 3 d, actively initiated chemotaxis and upregulated mRNA and protein immunomodulatory markers, particularly inducible nitric oxide synthase and toll-like receptor-4 up to 6 d, yet without a cytotoxic effect. Increased vascularization was demonstrated morphologically, preceded by high expression of vascular endothelial growth factor. The transition to appropriate tissue repair coincided with implant surface enrichment of Ca and P and reduced peri-implant Mg2+ concentration. Mg implants revealed a thinner fibrous encapsulation compared with Ti. The detailed understanding of the relationship between Mg material properties and the spatial and time-resolved cellular processes provides a basis for the interpretation of clinical observations and future tailoring of Mg implants.
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| 7. |
- Ben Amara, Heithem, 1984, et al.
(författare)
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Promoting soft and hard tissue repair via immunomodulation by the surface degradation of magnesium implants in vivo
- 2023
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Ingår i: Materials for Tomorrow conference by Chalmers University of Technology, 8-10 November 2023, Gothenburg, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: Magnesium (Mg) is a reactive metallic biomaterial that degrades via surface corrosion upon contact with body fluids. By virtue of its degradation and mechanical properties, Mg implants are currently employed with success to treat musculoskeletal injuries and avoid second-stage surgical removal 1. While these implants are claimed to possess anti-inflammatory properties, this notion contrasts with the initial signs of inflammation observed in the soft tissue of patients treated with Mg implants. This study investigated how the surface degradation of Mg implants in vivo influences the molecular, cellular, and structural events during initial inflammation and subsequent healing of the interfacing soft tissue and bone in comparison to nondegradable titanium (Ti) implants using experimental rat models. METHODS: Rats received disc-shaped implants in their dorsum subcutis or screw-shaped implants in the proximal metaphysis of their tibiae. Implants were manufactured from pure Mg (>99.995% - high purity) or from pure Ti (grade 4). Animals were euthanized after 1, 3, 6, 14, and 28 days of soft tissue implantation, and after 3 and 28 days of bone implantation. Two types of samples were collected: i) Implants only (n = 7-8/group/time-point): for counting and/or gene expression analyses of implant-adherent cells. ii) Implants with peri-implant tissues (n = 5-8/group/time-point): for compositional analysis of the Mg degradation layer in conjunction with the histomorphometry of the fibrous capsule around implants in soft tissues and of osseointegration at the bone–implant interface. Statistical comparisons were run using Kruskal-Wallis and Mann-Whitney tests (p<0.05). RESULTS: Cells adherent to the implant surfaces featured different gene regulation patterns between Mg and Ti groups (Fig. 1). Initially in soft tissue (1–6 d) and bone (3 d), a higher expression of proinflammatory macrophage polarization markers, e.g. inducible nitric oxide synthase (iNos), was shown in Mg versus Ti groups. Afterward, by 28 d, gene expression of both macrophage subtype markers (proinflammatory – iNos, and prohealing – Mannose receptor c1; Mrc1) was comparable between implants, irrespective of their insertion site. Histomorphometry revealed superior bone–implant contact (at 28 d in bone) and thinner fibrous capsule (at 6–28 d in soft tissue) for Mg versus Ti (Fig. 1). The 28 d-degradation layer at the Mg surface was enriched in Ca and P in both soft tissue and bone. CONCLUSIONS: In comparison to Ti implants, both soft tissue and bone responses to Mg implants featured an initial, amplified, yet transient, inflammation marked by the gene activation of the macrophage proinflammatory subtype. Such immunomodulation by the surface degradation of Mg implant promoted more bone deposition, at the bone–implant interface, and less fibrous encapsulation, at the soft tissue–implant interface. REFERENCES: 1. Han et al. Mater Today 2019, 23: 57-71. ACKNOWLEDGEMENTS: Horizon 2020 Marie Skłodowska-Curie Action (No 811226) and Area of Advance Materials/Chalmers and GU Biomaterials. Mg implants were generously provided by Hereon, Geesthacht, Germany.
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| 8. |
- Ben Amara, Heithem, 1984, et al.
(författare)
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Toward a disruptive, minimally invasive small finger joint implant concept: Cellular and molecular interactions with materials in vivo
- 2024
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Ingår i: ACTA BIOMATERIALIA. - 1742-7061 .- 1878-7568. ; 183, s. 130-145
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Tidskriftsartikel (refereegranskat)abstract
- Osteoarthritis (OA) poses significant therapeutic challenges, particularly OA that affects the hand. Currently available treatment strategies are often limited in terms of their efficacy in managing pain, regulating invasiveness, and restoring joint function. The APRICOT((R)) implant system developed by Aurora Medical Ltd (Chichester, UK) introduces a minimally invasive, bone-conserving approach for treating hand OA ( https://apricot-project.eu/ ). By utilizing polycarbonate urethane (PCU), this implant incorporates a caterpillar track-inspired design to promote the restoration of natural movement to the joint. Surface modifications of PCU have been proposed for the biological fixation of the implant. This study investigated the biocompatibility of PCU alone or in combination with two surface modifications, namely dopaminecarboxymethylcellulose (dCMC) and calcium-phosphate (CaP) coatings. In a rat soft tissue model, native and CaP-coated PCU foils did not increase cellular migration or cytotoxicity at the implant-soft tissue interface after 3 d, showing gene expression of proinflammatory cytokines similar to that in non-implanted sham sites. However, dCMC induced an amplified initial inflammatory response that was characterized by increased chemotaxis and cytotoxicity, as well as pronounced gene activation of proinflammatory macrophages and neoangiogenesis. By 21 d, inflammation subsided in all the groups, allowing for implant encapsulation. In a rat bone model, 6 d and 28 d after release of the periosteum, all implant types were adapted to the bone surface with a surrounding fibrous capsule and no protracted inflammatory response was observed. These findings demonstrated the biocompatibility of native and CaP-coated PCU foils as components of APRICOT((R)) implants.
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| 10. |
- Gerner, Erik, 1986, et al.
(författare)
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Targeting Pseudomonas aeruginosa quorum sensing with sodium salicylate modulates immune responses in vitro and in vivo.
- 2023
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Ingår i: Frontiers in cellular and infection microbiology. - 2235-2988. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Chronic infections are a major clinical challenge in hard-to-heal wounds and implanted devices. Pseudomonas aeruginosa is a common causative pathogen that produces numerous virulence factors. Due to the increasing problem of antibiotic resistance, new alternative treatment strategies are needed. Quorum sensing (QS) is a bacterial communication system that regulates virulence and dampens inflammation, promoting bacterial survival. QS inhibition is a potent strategy to reduce bacterial virulence and alleviate the negative impact on host immune response.This study investigates how secreted factors from P. aeruginosa PAO1, cultured in the presence or absence of the QS inhibitor sodium salicylate (NaSa), influence host immune response.In vitro, THP-1 macrophages and neutrophil-like HL-60 cells were used. In vivo, discs of titanium were implanted in a subcutaneous rat model with local administration of P. aeruginosa culture supernatants. The host immune response to virulence factors contained in culture supernatants (+/-NaSa) was characterized through cell viability, migration, phagocytosis, gene expression, cytokine secretion, and histology.In vitro, P. aeruginosa supernatants from NaSa-containing cultures significantly increased THP-1 phagocytosis and HL-60 cell migration compared with untreated supernatants (-NaSa). Stimulation with NaSa-treated supernatants in vivo resulted in: (i) significantly increased immune cell infiltration and cell attachment to titanium discs; (ii) increased gene expression of IL-8, IL-10, ARG1, and iNOS, and (iii) increased GRO-α protein secretion and decreased IL-1β, IL-6, and IL-1α secretion, as compared with untreated supernatants.In conclusion, treating P. aeruginosa with NaSa reduces the production of virulence factors and modulates major immune events, such as promoting phagocytosis and cell migration, and decreasing the secretion of several pro-inflammatory cytokines.
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