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Here, we present a detailed protocol for assessing the immunomodulatory potential of implant surfaces in vitro, aiming to improve the reliability and reproducibility of current protocols and promoting further research. Secretory cytokine profiles, mRNA expression, and cell surface markers were monitored using blood monocyte-derived macrophages to investigate macrophage polarization cultivated on titanium.
Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the first line of immune system interaction with implant surfaces, play a bidirectional role in modulating the inflammation-regeneration balance. For a deep understanding and the evaluation of the reactions between implant materials and immune responses, reliable in vitro methods and protocols are pivotal. Among different in vitro models, primary monocyte-derived macrophages (MDMs) present an excellent model for investigating macrophage-implant interactions. We have implemented an experimental protocol to evaluate the polarization of MDMs into M1 (classically activated) and M2 (alternatively activated) macrophages on implant surfaces. We isolated blood monocytes from healthy donors and differentiated them into macrophages using macrophage colony-stimulating factor (M-CSF). Differentiated macrophages were cultured on implant surfaces and polarized into M1 and M2 subtypes. M1 polarization was achieved in the presence of interferon (IFN)-γ and lipopolysaccharide (LPS), while M2 polarization was performed in a medium containing interleukin (IL)-4 and IL-13. We evaluated macrophage phenotypes by Enzyme-linked Immunosorbent Assay (ELISA), confocal laser scanning microscopy (CLSM), and quantitative real-time PCR (qRT-PCR) based on panels of secreted cytokines, cell surface markers, and expressed genes. The extracted RNA was transformed into complementary DNA (cDNA), and qRT-PCR was used to quantify mRNA related to M1 and M2 macrophages. Accordingly, M1 macrophages have been characterized by higher expression of proinflammatory Tumor necrosis factor (TNF-α) cytokine and CCR7 surface marker compared to M2 macrophages, which exhibited higher levels of CD209 and CCL13. Consequently, CCR7 and CD209 were identified as specific and reliable markers of M1 and M2 macrophage subtypes by immunostaining and visualizing by CLSM. Further confirmation was achieved by ELISA detecting elevated TNF-ɑ level in M1 and increased CCL13 in M2 cells. The proposed markers and experimental setup can be used effectively to assess the immunomodulatory potential of implants.
Implantable biomaterials have become a conventional solution for various human diseases and play a great role in biomedical research, including tissue engineering, drug delivery systems, and implants1,2. There is a wide range of implants made from various materials with different structures and functionalities, such as hip prostheses, stents, meshes, heart valves, or dental implants. Upon implantation, the tissue-implant contact provokes an immune response, followed by resolution, tissue remodeling, and homeostasis. These processes are influenced by the physical, chemical, and bioactive characteristics of the biomaterials used. These characteristics may affect the intensity and spectrum of pro- and anti-inflammatory responses, fibrotic capsule formation, tissue degradation, and healing phase3,4. In order to support and optimize the healing process and long-term implant integration, one emerging aspect of current research is to investigate and mediate the interaction between implant surfaces and immune cells.
Among other immune cells, macrophages, which are found throughout the body, are key players in inflammation and anti-pathogenic defense, as well as in healing processes and the maintenance of tissue homeostasis5,6. Based on their plasticity and the local tissue microenvironmental stimuli, macrophages are able to polarize into distinct functional phenotypes, which exhibit great differences in cell metabolism, cellular functions, and cytokine secretion profiles. Classically activated M1 phenotype can be distinguished by secretion of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, and is involved in the initial and chronic inflammatory response to trauma and foreign biomaterials. In contrast, alternatively, activated M2 macrophages, which are triggered by cytokines such as IL-4 and IL-13, have characteristic features like the resolution of inflammation and the promotion of tissue healing. M2-polarized macrophages can be identified by the expression of cell surface markers such as CD206 and the production of cytokines like IL-10 and IL-47. Similarly, macrophages that have already been polarized can reprogram themselves in a new microenvironment.
Many studies on cell-biomaterial interactions have shown the importance of macrophages in the cascade of immunologic responses toward implantable biomaterials and in orchestrating processes involved in the healing of implant-related complications8,9,10. Even though biomedical engineering has made significant progress in recent years, further research is needed to understand how implants modulate macrophage behavior and polarization11,12,13.
In cell culture, monocyte-derived peripheral blood mononuclear cells (PBMCs) can be differentiated into adherent M0 macrophages followed by induced polarization towards M1 or M2 phenotypes using LPS and IFN-γ or IL-4, respectively. After in vitro incubation with new biomaterial specimens, it is possible to utilize the different cell surface receptors and cytokine profiles of M1 and M2 macrophages to detect the immunomodulatory potential of biomaterials in vitro14,15. This study aimed to develop an in vitro protocol that can be employed to investigate the polarization of MDMs in response to different implant surfaces. Gene expression analyses, microscopy techniques, and ELISA can be used to determine the phenotypic markers and specific cytokine profiles of M1 and M2 macrophages modulated by the biomaterial. Hence, the complex interactions between macrophages and biomaterial surfaces can be elucidated, and valuable information can be obtained to better understand macrophage-biomaterial interactions. Finally, a standardized in vitro protocol ensures reproducibility, reliability, and comparability of experimental results by minimizing variability in the experimental setup.
Human peripheral blood was obtained from healthy blood donors in accordance with the protocol approved by the Ethics Committee of the medical faculty at the University of Tübingen (ethical approval: 286/2021 BO). Human PBMCs were isolated using Density Gradient Centrifugation, as previously described16. The following protocol is outlined for PBMCs isolated from 24 mL of blood. A schematic representation of the protocol is shown in Figure 1.
NOTE: Blood volume should be adjusted depending on the number of M0 macrophages used.
A total of 35.46 ± 9.1 million PBMCs were obtained from 24 mL of blood, resulting in 1.97 ± 0.46 million M0 macrophages (n = 5). All reagents, consumables, and devices are listed in the Table of Materials. Buffers are listed in Table 1.
1. Differentiation of human blood monocytes to macrophages
2. Cultivation and polarization of MDMs on the titanium implant surface
NOTE: On day 6 of differentiation, M0 macrophages were seeded on the biomaterial surfaces with different stimuli for 48 h to obtain fully polarized M1 or M2 macrophages. For each surface examined, three discs were used to seed M0, M1, and M2 macrophages. Cell culture-treated plastic coverslips were used as control surfaces.
3. Characterization of polarized macrophages using ELISA
NOTE: On day 2 of polarization, samples were prepared for characterization analyses. TNF-ɑ cytokine and CCL13 chemokine were measured to characterize M1 and M2 polarized macrophages, respectively. The concentration of secreted proteins was normalized to the total concentration of secreted proteins in the corresponding supernatant.
4. Characterization of polarized macrophages using CLSM
NOTE: Polarized macrophages were further characterized by staining them with antibodies to CD209 and CCR7 cell surface markers. The nuclei were counterstained with DRAQ5. CD68 or other markers can be used as pan-macrophage markers.
5. Characterization of polarized macrophages using qRT-PCR
NOTE: For RNA isolation, two discs per sample were used in order to obtain enough RNA for cDNA synthesis.
6. Statistical analysis
The results of this study demonstrate successful differentiation and polarization of MDMs on titanium surfaces, followed by characterization of M1 or M2 polarized macrophages. In the first step, we characterized polarized MDMs using CLSM. Based on our preliminary studies, CD209 and CCR7 were used as specific markers to differentiate M1 from M2 polarized MDMs. As shown in Figure 2A,B, MDMs polarized successfully into M1 and M2 macrophages. On the titanium surface, CCR7 was ex...
A thorough understanding of macrophage behavior is essential for comprehending the immunomodulatory properties of implantable materials. Several studies have reported heterogeneous markers, a variety of cell models, and protocols for characterizing macrophage polarization in vitro17,18,19,20. To improve reproducibility, reliability, and comparability of experimental results, standardiz...
The authors have nothing to disclose.
The discs were kindly provided by Medentis Medical, Bad-Neuenahr-Ahrweiler, Germany. The authors acknowledge the support from the Department of Oral and Maxillofacial Surgery (University Hospital Tuebingen).
Name | Company | Catalog Number | Comments |
24-well plate, not-treated | Corning Incorporated (Kennebunk, USA) | 144530 | |
Absorbance reader Infinite F50 | TECAN Austria GmbH (Grödig, Austria) | TCAT91000001 | |
Accutase in DPBS, 0.5 mM EDTA | EMD Millipore Corp. (Burlington, USA) | SCR005 | |
Anti-Fade Fluorescence Mounting Medium -Aqueous, Fluoroshield | abcam (Cambridge, UK) | ab104135 | |
Bio-Rad MJ Research PTC-200 Peltier Thermal Cycler | Bio-Rad / MJ Research | 7212 | |
Bovine serum albumin (BSA) | VWR International bvba (Leuven, Belgium) | 422361V | |
Centrifuge 5804 R | Eppendorf SE (Hamburg, Germany) | 5804 R | |
DC-SIGN (D7F5C) XP Rabbit mAb | Cell Signaling Technology | 13193 | |
Dimethyl sulfoxide | Sigma Aldrich Co. (St.Louis, MO, USA) | D2438-5X10ML | |
DRAQ5 Staining Solution | Milteny Biotec (Bergisch Gladbach, Germany) | 130-117-344 | |
Ethanol ≥99.8% for molecularbiology | Carl Roth GmbH + CO. KG (Karlsruhe, Germany) | 1HPH.1 | |
Goat Anti-Mouse IgG (H&L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 | Invitrogen | A32723TR | |
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Cyanine3 | Invitrogen | A10520 | |
GraphPad Prism | GraphPad | Version 9.4.1 | |
Human CCR7 Antibody | R&D Systems | MAB197 | |
Human IFN-gamma Recombinant | Invitrogen (Rockford, USA) | RIFNG100 | |
Human IL-13 | Milteny Biotec (Bergisch Gladbach, Germany) | 5230901032 | |
Human IL-4 | Milteny Biotec (Bergisch Gladbach, Germany) | 130-095-373 | |
Human M-CSF | Peprotech (Cranbury, USA) | 300-25 | |
Leica TCS SP5 | Leica Microsystems CMS GmbH (Mannheim, Germany) | https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp5/ | |
Lipopolysaccharides from Escherichia | Sigma Aldrich Co. (Missouri, USA) | L4391-1MG | |
Luna Universal qPCR Master Mix | New England Biolabs | NEB #M3003 | |
LunaScript RT SuperMix Kit | New England Biolabs | E3010L | |
Lymphocyte Separation Medium 1077 | PromoCell (Heidelberg, Germany) | C-44010 | |
MCP-4/CCL13 Human ELISA Kit | Invitrogen | EHCCL13 | |
MicroAmp Fast 96-Well Reaction Plate (0.1 mL) | Applied Biosystems (Waltham, USA) | 4346907 | |
MicroAmp Optical Adhesive Film | Life Technologies Corporation (Carlsbad, USA) | 4311971 | |
MicroAmp Splash Free 96-Well Base | Applied Biosystems (Waltham, USA) | 4312063 | |
Microlitercentrifuge CD-3124R | Phoenix Instrument (Germany) | 9013111121 | |
Microscope Cover Glasses, 10 mm | Carl Roth GmbH, Karlsruhe, Germany | 4HX4.1 | |
Monocyte Attachment Medium | PromoCell (Heidelberg, Germany) | C-28051 | |
Multiply-Pro Gefäß 0.5 mL, PP | Sarstedt AG & CO (Nümbrecht, Germany) | 7,27,35,100 | |
Nanodrop One | Thermo Scientific (USA) | ND-ONE-W | |
QuantStudio 3 System | Life Technologies GmbH (St. Leon-Rot, Germany) | A28567 | |
RNeasy Micro Kit | Qiagen | 74007 | |
RPMI 1640, 1x, with L-glutamine | Mediatech, Inc. (Manassas, USA) | 10-040-CV | |
Sterile bench, LaminarAir HB 2472 | Heraeus instruments (Hanau, Germany) | 51012197 | |
Tissue Culture Coverslips 13 mm (Plastic) | Sarstedt Inc. (Newton, USA) | 83,18,40,002 | |
Titanium machinied discs 12 cm | Medentis Medical (Bad-Neuenahr-Ahrweiler, Germany) | N/A | |
TNF alpha Human ELISA Kit | Invitrogen | KHC3011 | |
Trypan blue solution 0.4% | Carl Roth GmbH + Co. KG (Karlsruhe, Germany) | 1680.1 |
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