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.

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.

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 mm2). 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-ß1 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.

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.

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 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.