Generation of anti-inflammatory macrophages for implants and regenerative medicine using self-standing release systems with a phenotype-fixing cytokine cocktail formulation.
The immediate tissue microenvironment of implanted biomedical devices and engineered tissues is highly influential on their long term fate and efficacy. The creation of a long-term anti-inflammatory microenvironment around implants and artificial tissues can facilitate their integration. Macrophages are highly plastic cells that define the tissue reactions on the implanted material. Local control of macrophage phenotype by long-term fixation of their healing activities and suppression of inflammatory reactions are required to improve implant acceptance. Herein, we describe the development of a cytokine cocktail (M2Ct) that induces stable M2-like macrophage phenotype with significantly decreased pro-inflammatory cytokine and increased anti-inflammatory cytokine secretion profile. The positive effect of the M2Ct was shown in an in vitro wound healing model; where M2Ct facilitated wound closure by human fibroblasts in co-culture conditions. Using a model for induction of inflammation by LPS we have shown that the M2Ct phenotype is stable for 12days. However, in the absence of M2Ct in the medium macrophages underwent rapid pro-inflammatory re-programming upon IFNg stimulation. Therefore, loading and release of the cytokine cocktail from a self-standing, transferable gelatin/tyraminated hyaluronic acid based release system was developed to stabilize macrophage phenotype for in vivo applications in implantation and tissue engineering. The M2Ct cytokine cocktail retained its anti-inflammatory activity in controlled release conditions. Our data indicate that the direct application of a potent M2 inducing cytokine cocktail in a transferable release system can significantly improve the long term functionality of biomedical devices by decreasing pro-inflammatory cytokine secretion and increasing the rate of wound healing.



Uncontrollable activation of macrophages in the microenvironment of implants and engineered tissues is a significant problem leading to poor integration of implants and artificial tissues. In the current manuscript we demonstrate that self-standing, transferable gelatin/tyraminated hyaluronic acid based thin films are perspective tools for controlled release of anti-inflammatory cytokine combinations and can be used to down-modulate macrophage activation on implant surfaces. We also show that optimized cytokine cocktail consisting of IL4/IL10/TGFβ1 (M2Ct) induces long-term anti-inflammatory and pro-healing phenotype in human primary monocyte-derived macrophages. This cocktail formulation could be loaded on gelatin/tyraminated films and promoted favorable M2-like macrophage phenotype with low responsiveness to pro-inflammatory stimuli. Such self-standing release systems can be used for prolonged local control of macrophage phenotype upon implantation.
Institute for Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany; Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, 36 Lenin Prospekt, Tomsk 634050, Russia.
Vladimir
V
Riabov
Division of Immunology, Queen's Medical Centre, School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
Fabián
F
Salazar
Division of Immunology, Queen's Medical Centre, School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
Su Su
SS
Htwe
Institute for Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany.
Alexandru
A
Gudima
Institute for Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany.
Christina
C
Schmuttermaier
Protip Medical, 8 Place de l'Hopital, 67000 Strasbourg, France.
Julien
J
Barthes
INSERM UMR 1121, Biomaterials and Bioengineering, 11 rue Humann, 67000 Strasbourg, France; Faculté de Chirurgie Dentaire, Université de Strasbourg, 3 rue Sainte Elisabeth, 67000 Strasbourg, France.
Helena
H
Knopf-Marques
Institute for Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany; Red Cross Blood Service Baden-Württemberg-Hessen, Friedrich-Ebert Str. 107, D-68167 Mannheim, Germany.
Harald
H
Klüter
Division of Immunology, Queen's Medical Centre, School of Life Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
Amir M
AM
Ghaemmaghami
Protip Medical, 8 Place de l'Hopital, 67000 Strasbourg, France; INSERM UMR 1121, Biomaterials and Bioengineering, 11 rue Humann, 67000 Strasbourg, France.
Nihal Engin
NE
Vrana
Institute for Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany; Red Cross Blood Service Baden-Württemberg-Hessen, Friedrich-Ebert Str. 107, D-68167 Mannheim, Germany; Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, 36 Lenin Prospekt, Tomsk 634050, Russia. Electronic address: julia.kzhyshkowska@medma.uni-heidelberg.de.
Julia
J
Kzhyshkowska
D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism; D016207:Cytokines / Q000276:immunology*; D003692:Delayed-Action Preparations / Q000138:chemical synthesis; D006801:Humans; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000637:transplantation*; D019736:Prostheses and Implants; D012038:Regeneration / Q000276:immunology*; D054457:Tissue Scaffolds; D014945:Wound Healing / Q000276:immunology*
D003470:Culture Media; D016207:Cytokines; D003692:Delayed-Action Preparations
Controlled release; Cytokine; Gelatin; Macrophage phenotype control; Wound healing