Impact of a High-Fat Diet at a Young Age on Wound Healing in Mice

As the prevalence of juvenile-onset obesity rises globally, the multitude of related health consequences gain significant importance. In this context, obesity is associated with impaired cutaneous wound healing. In experimental settings, mice are the most frequently used model for investigating the effect of high-fat diet (HFD) chow on wound healing in wild-type or genetically manipulated animals, e.g., diabetic ob/ob and db/db mice. However, these studies have mainly been performed on adult animals. Thus, in the present study, we introduced a mouse model for a juvenile onset of obesity. We exposed 4-week-old mice to an investigational feeding period of 9 weeks with an HFD compared to a regular diet (RD). At a mouse age of 13 weeks, we performed excisional and incisional wounding and measured the healing rate. Wound healing was examined by serial photographs with daily wound size measurements of the excisional wounds. Histology from incisional wounds was performed to quantify granulation tissue (thickness, quality) and angiogenesis (number of blood vessels per mm$^{2}$). The expression of extracellular matrix proteins (collagen types I/III/IV, fibronectin 1, elastin), inflammatory cytokines (MIF, MIF-2, IL-6, TNF-α), myofibroblast differentiation (α-SMA) and macrophage polarization (CD11c, CD301b) in the incisional wounds were evaluated by RT-qPCR and by immunohistochemistry. There was a marked delay of wound closure in the HFD group with a decrease in granulation tissue quality and thickness. Additionally, inflammatory cytokines (MIF, IL-6, TNF-α) were significantly up-regulated in HFD- when compared to RD-fed mice measured at day 3. By contrast, MIF-2 and blood vessel expression were significantly reduced in the HFD animals, starting at day 1. No significant changes were observed in macrophage polarization, collagen expression, and levels of TGF-β1 and PDGF-A. Our findings support that an early exposition to HFD resulted in juvenile obesity in mice with impaired wound repair mechanisms, which may be used as a murine model for obesity-related studies in the future

Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p < 0.01) when compared to mSVF alone. Also, GelMA-mSVF secreted stable levels of bFGF over 21 days. While VEGF-A was primarily expressed on day 21, perilipin-2 and CD73-positive cells were observed on days 7 and 21. Finally, GelMA-mSVF significantly improved fibroblast viability as compared with GelMA alone (p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation

Murine iPSC-Loaded Scaffold Grafts Improve Bone Regeneration in Critical-Size Bone Defects

In certain situations, bones do not heal completely after fracturing. One of these situations is a critical-size bone defect where the bone cannot heal spontaneously. In such a case, complex fracture treatment over a long period of time is required, which carries a relevant risk of complications. The common methods used, such as autologous and allogeneic grafts, do not always lead to successful treatment results. Current approaches to increasing bone formation to bridge the gap include the application of stem cells on the fracture side. While most studies investigated the use of mesenchymal stromal cells, less evidence exists about induced pluripotent stem cells (iPSC). In this study, we investigated the potential of mouse iPSC-loaded scaffolds and decellularized scaffolds containing extracellular matrix from iPSCs for treating critical-size bone defects in a mouse model. In vitro differentiation followed by Alizarin Red staining and quantitative reverse transcription polymerase chain reaction confirmed the osteogenic differentiation potential of the iPSCs lines. Subsequently, an in vivo trial using a mouse model (n = 12) for critical-size bone defect was conducted, in which a PLGA/aCaP osteoconductive scaffold was transplanted into the bone defect for 9 weeks. Three groups (each n = 4) were defined as (1) osteoconductive scaffold only (control), (2) iPSC-derived extracellular matrix seeded on a scaffold and (3) iPSC seeded on a scaffold. Micro-CT and histological analysis show that iPSCs grafted onto an osteoconductive scaffold followed by induction of osteogenic differentiation resulted in significantly higher bone volume 9 weeks after implantation than an osteoconductive scaffold alone. Transplantation of iPSC-seeded PLGA/aCaP scaffolds may improve bone regeneration in critical-size bone defects in mice

SAXS imaging reveals optimized osseointegration properties of bioengineered oriented 3D-PLGA/aCaP scaffolds in a critical size bone defect model

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals

The role of MIF-2 in obesity-induced wound healing disorder

Wounds that do not undergo sufficient healing or even become chronic wounds represent a major challenge for medicine. Obese individuals are prone to non-healing wounds. In this context, the adipose tissue can become chronically inflamed, causing a pathological alteration at the molecular and cellular level. This results in impaired cell migration, proliferation and differentiation, as well as a reduced angiogenesis. Since the adipose tissue is located in immediate vicinity of cutaneous wounds and is involved in wound healing, the obesity-induced cellular and molecular changes also negatively influence the wound healing process. Although obesity-induced chronic wounds have been studied for a long time and a lot is known, there our understanding of the mechanisms is still limited. One pathologically altered protein family is the macrophage migration inhibitor factor (MIF) family, in human encompassing MIF and MIF-2. While MIF has been well studied, MIF 2 was only recently discovered as a homologue of MIF. Both bind to the same receptor, CD74, while only MIF interacts with CXCR2, and they differ in various systemic conditions e.g. in obesity, in which MIF is increased and MIF 2 is decreased. In inflammation, MIF polarizes macrophages towards a pro-inflammatory M1-like phenotype and MIF-2 towards an anti-inflammatory M2-like subtype. The role of MIF in wound healing is still controversially discussed, but it seems that its function differs in distinct phases and tissue types. MIF-2 was recently reported to exhibit a positive effect on wound healing. Thus, in this thesis, the role of MIF-2 in wound healing was investigated in a murine wound healing model employing Mif-2–/– mice, accompanied by mechanistic in vitro experiments. Both mice groups, the obese wild type as well as the Mif-2–/– mice showed a delayed wound healing when compared to the control lean wild type mice. Their wound sizes became even wider during the first days. Immunohistochemical analysis confirmed the impaired healing process in obese and Mif-2–/– mice. Furthermore, tissue examinations and genetic analysis showed reduced collagen production and granulation tissue formation as well as a diminished myofibroblast differentiation, which was most pronounced in the Mif-2–/– mice. In in vitro experiments, NIH/3T3 cells were differentiated into myofibroblasts when treated with recombinant MIF 2 or a MIF 2 inhibitor. When MIF 2 was inhibited, differentiation potential decreased sharply even at low doses. At higher concentrations of recombinant MIF 2, cell differentiation was also partially reduced. My experiments revealed that MIF-2 is crucial for the differentiation from “wound fibroblasts” into myofibroblasts, which are primarily responsible for collagen production and formation of granulation tissue. In an in vivo wound model characterized by obese and Mif 2–/– mice as well as applying in vitro pathway analyses, the deletion of MIF-2 resulted in an impaired differentiation and wound closure. It appeared that MIF-2 influenced the canonical TGF-β/Smad pathway, the main pathway for myofibroblasts differentiation, via an ERK1/2-MAPK downstream cascade and a so far unknown key protein. However, I also obtained evidence in my thesis that an excess of MIF-2 leads to a partial inhibition of the differentiation, most likely also via ERK1/2, suggestive of a differential dose behavior. In conclusion, the obesity-induced downregulation of MIF 2 seems to be one major reason for a delayed and insufficient wound healing process in obese individuals. Furthermore, my results indicates that this impairment is caused by diminished myofibroblast differentiation resulting in delayed granulation tissue formation.Nicht ausreichend heilende Wunden und chronische Wunden stellen eine große Herausforderung für die Medizin dar. Übergewichtige Personen sind anfälliger für nicht heilende Wunden. Ihr Fettgewebe ist chronisch entzündet, was eine pathologische Veränderung auf molekularer und zellulärer Ebene zur Folge hat. Dies führt zu einer beeinträchtigten Zellmigration, -proliferation und -differenzierung sowie einer verminderten Angiogenese. Da sich das Fettgewebe in unmittelbarer Nähe zu kutanen Wunden befindet und an der Wundheilung beteiligt ist, beeinflussen durch Fettleibigkeit bedingte zelluläre und molekulare Veränderungen auch die Wundheilung negativ. Obwohl Adipositas-induzierte chronische Wunden seit langem untersucht werden und bereits einiges darüber bekannt ist, fehlt noch viel, um den gesamten Mechanismus zu verstehen. Eine pathologisch veränderte Protein Gruppe ist die Makrophagen Migration Inhibition Faktor (MIF) Familie, MIF und MIF-2. Während MIF seit langem gut erforscht ist, wurde MIF-2 erst kürzlich als MIF-Homolog entdeckt. Beide binden an denselben Rezeptor, CD74, unterscheiden sich jedoch in verschiedenen systemischen Zuständen wie bei Fettleibigkeit, MIF ist erhöht und MIF-2 erniedrigt. Bei Entzündungen polarisiert MIF Makrophagen zum pro-inflammatorischen M1 und MIF-2 zum anti-inflammatorischen M2 Phänotypen. Die Rolle von MIF in der Wundheilung wird noch kontrovers diskutiert, aber seine Funktion scheint sich in verschiedenen Phasen und Gewebetypen zu unterscheiden. Kürzlich gab es Hinweise für einen positiven Effekt von MIF-2 auf die Wundheilung. Daher wurde in dieser Arbeit die Rolle von MIF-2 bei der Wundheilung in einem murinen Wundheilungsmodel mit Mif-2–/– Mäusen untersucht und mit in-vitro Experimenten zum Mechanismus untermauert. Sowohl die adipösen Wildtypen als auch die Mif-2–/– Mäuse wiesen eine verzögerte Wundheilung auf im Vergleich zu den normalgewichtigen Kontrollmäusen. Während den ersten Tagen hat sich das Wundareal sogar erweitert. Immunhistologische Analysen bestätigten den verschlechterten Heilungsprozess in der adipösen und in der Mif-2–/– Gruppe. Zusätzlich wiesen Gewebeanalysen und genetische Untersuchungen eine reduzierte Produktion von Kollagen und des Granulationsgewebes auf, sowie eine verminderte Myofibroblastendifferenzierung, welche am stärksten bei den Mif-2–/– Mäusen vorhanden war. NIH/3T3 Zellen wurden in in-vitro Experimenten zu Myofibroblasten differenziert unter der Behandlung von rekombinantem MIF-2 oder einem MIF-2 Inhibitor. Wenn die Zellen mit dem MIF-2 Inhibitor behandelt wurden, sank das Differenzierungspotential bereits bei geringer Konzentration rapide ab. Ebenso hatten hohe Dosen vom rekombinantem MIF-2 eine partielle Reduzierung der Differenzierbarkeit zufolge In meiner Arbeit konnte ich zeigen, dass MIF-2 eine zentrale Rolle für die Differenzierung von “Wund-Fibroblasten” zu Myofibroblasten hat, welche hauptverantwortlich sind für die Kollagenproduktion und für die Bildung des Granulationsgewebes. Sowohl in dem in-vivo Wundheilungsmodel als auch in den in-vitro Mechanismusanalysen führte die Deletion von MIF-2 zu einer beeinträchtigen Differenzierung und Wundschliessung. Es scheint, dass MIF-2 den kanonischen TGF β/Smad-Weg, den Hauptweg für die Differenzierung von Myofibroblasten, über die ERK1/2-MAPK-Downstream-Kaskade beeinflusst und ein bisher unbekanntes Schlüsselprotein ist. Allerdings wurde in der Dissertation auch gezeigt, dass ein Überschuss an MIF-2 auch zu einer teilweisen Hemmung der Differenzierung führt, höchstwahrscheinlich auch über ERK1/2. Zusammengefasst scheint die durch Fettleibigkeit induzierte Herunterregulation von MIF-2 ein Hauptfaktor für die verzögerte und unzureichende Wundheilung zu sein, welche bei Adipositas beobachtet werden kann. Weiterhin geben meine Ergebnisse Hinweise darauf, dass die Beeinträchtigung durch eine verminderte Differenzierung von Myofibroblasten herführt, wodurch die Bildung von Granulationsgewebe verzögert wird

Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation

Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p < 0.01) when compared to mSVF alone. Also, GelMA-mSVF secreted stable levels of bFGF over 21 days. While VEGF-A was primarily expressed on day 21, perilipin-2 and CD73-positive cells were observed on days 7 and 21. Finally, GelMA-mSVF significantly improved fibroblast viability as compared with GelMA alone (p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation.ISSN:1422-006

The effect of the macrophage migration inhibitory factor (MIF) on excisional wound healing in vivo

Background: The macrophage migration inhibitory factor (MIF) has been determined as a cytokine exerting a multitude of effects in inflammation and angiogenesis. Earlier studies have indicated that MIF may also be involved in wound healing and flap surgery. Methods: We investigated the effect of MIF in an excisional wound model in wildtype, Mif-/- and recombinant MIF treated mice. Wound closure rates as well as the macrophage marker Mac-3, the pro-inflammatory cytokine tumor necrosis factor α (TNFα) and the pro-angiogenic factor von Willebrand factor (vWF) were measured. Finally, we used a flap model in Mif-/- and WT mice with an established perfusion gradient to identify MIF's contribution in flap perfusion. Results: In the excision wound model, we found reduced wound healing after MIF injection, whereas Mif deletion improved wound healing. Furthermore, a reduced expression of Mac-3, TNFα and vWF in Mif-/- mice was seen when compared to WT mice. In the flap model, Mif-/- knockout mice showed mitigated flap perfusion with lower hemoglobin content and oxygen saturation as measured by O2C measurements when compared to WT mice. Conclusions: Our data suggest an inhibiting effect of MIF in wound healing with increased inflammation and perfusion. In flaps, by contrast, MIF may contribute to flap vascularization

Murine iPSC-Loaded Scaffold Grafts Improve Bone Regeneration in Critical-Size Bone Defects

In certain situations, bones do not heal completely after fracturing. One of these situations is a critical-size bone defect where the bone cannot heal spontaneously. In such a case, complex fracture treatment over a long period of time is required, which carries a relevant risk of complications. The common methods used, such as autologous and allogeneic grafts, do not always lead to successful treatment results. Current approaches to increasing bone formation to bridge the gap include the application of stem cells on the fracture side. While most studies investigated the use of mesenchymal stromal cells, less evidence exists about induced pluripotent stem cells (iPSC). In this study, we investigated the potential of mouse iPSC-loaded scaffolds and decellularized scaffolds containing extracellular matrix from iPSCs for treating critical-size bone defects in a mouse model. In vitro differentiation followed by Alizarin Red staining and quantitative reverse transcription polymerase chain reaction confirmed the osteogenic differentiation potential of the iPSCs lines. Subsequently, an in vivo trial using a mouse model (n = 12) for critical-size bone defect was conducted, in which a PLGA/aCaP osteoconductive scaffold was transplanted into the bone defect for 9 weeks. Three groups (each n = 4) were defined as (1) osteoconductive scaffold only (control), (2) iPSC-derived extracellular matrix seeded on a scaffold and (3) iPSC seeded on a scaffold. Micro-CT and histological analysis show that iPSCs grafted onto an osteoconductive scaffold followed by induction of osteogenic differentiation resulted in significantly higher bone volume 9 weeks after implantation than an osteoconductive scaffold alone. Transplantation of iPSC-seeded PLGA/aCaP scaffolds may improve bone regeneration in critical-size bone defects in mice.ISSN:1422-006

Differential regulation of macrophage activation by the MIF cytokine superfamily members MIF and MIF-2 in adipose tissue during endotoxemia

Sepsis is a leading cause of death worldwide and recent studies have shown white adipose tissue (WAT) to be an important regulator in septic conditions. In the present study, the role of the inflammatory cytokine macrophage migration inhibitory factor (MIF) and its structural homolog D-dopachrome tautomerase (D-DT/MIF-2) were investigated in WAT in a murine endotoxemia model. Both MIF and MIF-2 levels were increased in the peritoneal fluid of LPS-challenged wild-type mice, yet, in visceral WAT, the proteins were differentially regulated, with elevated MIF but downregulated MIF-2 expression in adipocytes. Mif gene deletion polarized adipose tissue macrophages (ATM) toward an anti-inflammatory phenotype while Mif-2 gene knockout drove ATMs toward a pro-inflammatory phenotype and Mif-deficiency was found to increase fibroblast viability. Additionally, we observed the same differential regulation of these two MIF family proteins in human adipose tissue in septic vs healthy patients. Taken together, these data suggest an inverse relationship between adipocyte MIF and MIF-2 expression during systemic inflammation, with the downregulation of MIF-2 in fat tissue potentially increasing pro-inflammatory macrophage polarization to further drive adipose inflammation