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.

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.

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-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.

Large bone defects remain a major clinical challenge, as current treatments primarily provide mechanical stability while often insufficiently addressing the biological microenvironment. The cell-deposited extracellular matrix (CD-ECM) represents a promising strategy to improve implant bioactivity by mimicking key features of the native tissue. In this study, we compared CD-ECMs from adipose tissue-derived mesenchymal stromal cells (ASCs), ASC-derived osteoprogenitor cells, and dermal fibroblasts. ECM composition was analyzed, and its ability to support the osteogenesis of reseeded skeletal stem cells (SSCs) was assessed. Subsequently, the best performing cells were used to produce CD-ECM on a 3D scaffold. Furthermore, we improved the ECM by treating the ECM-producing cells with dextran sulfate (Dx-S). Fibroblast-derived ECM showed higher collagen and glycosaminoglycan contents compared to ASC-ECM or osteoprogenitor-ECM. Furthermore, only the fibroblast-derived ECM (Fibro-ECM) exerted a supportive effect on the osteogenesis of SSCs. SSCs seeded on ECM showed a higher proliferation rate and enhanced osteogenesis. Supplementation with dextran sulfate further increased ECM deposition and osteogenic potential. We showed that fibroblasts produced substantially more ECM with a stronger pro-osteogenic effect than ASCs or osteoprogenitor cells. The ECM and its pro-osteogenic effect could further be increased when fibroblasts were treated with Dx-S. Together, these results highlight Fibro-ECM as a promising and easily accessible cell-derived ECM deposition strategy to improve the biological performance of implants in bone regeneration.

Critical size bone defects represent a clinical challenge, associated with considerable morbidity, and frequently trigger the requirement of secondary procedure. To fill osseous gaps, multiple steps are required, such as proliferation and differentiation on the cellular level and the building of extracellular matrix. In addition, the osteogenic potential of cell-derived extracellular matrices (CD-ECM) is known to enhance bone healing. We therefore examined the osteogenic potential of fibroblast-derived ECM (Fibro-ECM) and assessed the influence of Dextran-sulfate (Dx-S) addition regarding the production of extracellular matrix (ECM). ECMs were generated by culturing human dermal fibroblasts, adipose-derived stromal cells (ASCs), and osteoblasts derived from ASCs (Osteo-ECM) for four days, with or without Dx-S supplementation. After decellularization, skeletal stem cells (SSCs) isolated from femoral head aspirations were seeded onto the ECMs and differentiated under osteogenic conditions for 17 days. Osteogenesis was assessed by Alizarin Red S staining for calcium deposition and RT-qPCR analysis of osteogenic marker genes. SSCs cultured on Fibro-ECM exhibited enhanced osteogenesis compared to Osteo-ECM and ASC-derived ECM, as evidenced by increased calcium deposition. Notably, Dx-S supplementation further improved the osteoinductive capacity of Fibro-ECM, leading to an upregulation of osteocalcin (OCN) and bone morphogenetic protein 2 (BMP2). In contrast, Dx-S had no significant effect on Osteo-ECM. The addition of Dx-S in autologous fibroblast-derived ECM induces an improvement in osteoinductivity. Addition of Dx-S may therefore be a useful adjunct in the in vitro bone generation models. Whether these results may represent a piece in the puzzle for difficult healing situations in patients with nonunions and bone defects should be subject to further study.