TLR4-dependent shaping of the wound site by MSCs accelerates wound healing.

We here address the question whether the unique capacity of mesenchymal stem cells to re-establish tissue homeostasis depends on their potential to sense pathogen-associated molecular pattern and, in consequence, mount an adaptive response in the interest of tissue repair. After injection of MSCs primed with the bacterial wall component LPS into murine wounds, an unexpected acceleration of healing occurs, clearly exceeding that of non-primed MSCs. This correlates with a fundamental reprogramming of the transcriptome in LPS-treated MSCs as deduced from RNAseq analysis and its validation. A network of genes mediating the adaptive response through the Toll-like receptor 4 (TLR4) pathway responsible for neutrophil and macrophage recruitment and their activation profoundly contributes to enhanced wound healing. In fact, injection of LPS-primed MSCs silenced for TLR4 fails to accelerate wound healing. These unprecedented findings hold substantial promise to refine current MSC-based therapies for difficult-to-treat wounds.

Biomechanical and histological analyses of the fracture healing process after direct or prolonged reduction.

BACKGROUND: Reduction of femoral shaft fractures remains a challenging problem in orthopaedic surgery. Robot-assisted reduction might ease reduction and fracture treatment. However, the influence of different reduction pathways on patients' physiology is not fully known yet. Therefore, the aim of this study was to examine the biomechanics and histology of fracture healing after direct and prolonged robot-assisted reduction in an in vivo rat model. METHODS: 144 male CD® rats were randomly assigned to 12 groups. Each animal received an external fixator and an osteotomy on the left femoral shaft. On the fourth postoperative day, the 1× reduction groups received a single reduction maneuver, whereas the 10× reduction groups received the same reduction pathway with ten repetitions. The control groups did not undergo any reduction maneuvers. Animals were killed after 1, 2, 3 and 4 weeks, respectively, and the composition of the fracture gap was analyzed by µCT and non-decalcified histology. Biomechanical properties were investigated by a three-point bending test, and the bone turnover markers PINP, bCTx, OPG, sRANKL, TRACP-5b, BALP, and OT/BGP were measured. RESULTS: One week after the reduction maneuver, µCT analysis showed a higher cortical bone volume in the 1× reduction group compared to the 10× reduction group. Biomechanically, the control group showed higher maximum force values measured by three-point bending test compared to both reduction groups. Furthermore, less collagen I formation was examined in the 10× reduction group compared to the control group after 1 week of fracture healing. PINP concentration was decreased in 10× reduction group after 1 week compared to control group. The same trend was seen after 3 weeks. CONCLUSION: A single reduction maneuver has a beneficial effect in the early phase of the fracture healing process compared to repeated reduction maneuvers. In the later phase of fracture healing, no differences were found between the groups.

Repetitive reduction lead to significant elevated IL-6 and decreased IL-10 levels in femoral osteotomies: A quantitative analysis of a robot-assisted reduction process in a rat model.

INTRODUCTION: The field of robot-assisted fracture reduction has been developed by several research groups over more than one decade by now, with the main goals of increasing the fracture reduction accuracy. However, the influence of different reduction paths to patients' physiology is not fully known yet. The aim of our study was to compare the impacts of a robot-assisted direct reduction path versus an artificially prolonged reduction path by measuring the cytokine responses in an in vivo rat model. MATERIALS AND METHODS: Thirty-six male CD(©) rats were assigned into three groups with an external fixator and osteotomy on the left femur. Seven days later, the robot was attached and one group was reduced in a single attempt, while the other group underwent 10 attempts by the robot. The third group was the control group without reduction. Before, and as well as 6, 24 and 48h after the reduction process blood samples were collected. IL-1, IL-6, IL-10, IL-17, and MCP-1 concentrations where analysed via ELISA or cytometric bead assay. Muscle biopsies in the osteotomy area were collected 48h after the reduction process for histological analyses. Statistical significance was set at p≤0.05. RESULTS: Analysis of the cytokines showed that the pro-inflammatory cytokine IL-6 of the Ten-Attempts reduction group significantly increased 6h after reduction compared to the control group. IL-6 further showed markedly elevated levels 6h after surgery in the Ten-Attempts reduction group compared to the Single-Attempt reduction group. On the anti-inflammatory side, IL-10 showed a significant decrease in the Ten-Attempts reduction group 6h after reduction compared to the Single-Attempt reduction and control group. Muscle biopsies showed a significant increase of pathological changes in both reduction groups and an increase in the severity of bleedings of the Ten-Attempts reduction group compared to the Single-Attempt reduction and control group. CONCLUSION: A direct and gentle reduction procedure as feasible by the aid of a robot is preferable over a prolonged reduction in terms of cytokine response and tissue changes.

Peripheral but not central leptin treatment increases numbers of circulating NK cells, granulocytes and specific monocyte subpopulations in non-endotoxaemic lean and obese LEW-rats.

Leptin, a hormone mainly generated by adipocytes, acts centrally in the hypothalamus to regulate body weight and energy expenditure. However, there is strong evidence that leptin is also involved in cell-mediated immunity and cytokine crosstalk. In the present study the effects of diet-induced obesity and central and peripheral leptin treatment on leukocyte subsets and cytokine production was investigated. Leptin was injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) in male endotoxaemic or vehicle-treated healthy LEW-rats. Numbers of blood leukocyte subsets were analysed by FACS and cytokines (TNF-alpha and IL-6) by ELISA. Results showed that peripheral rather than central leptin treatment was able to significantly increase numbers of granulocytes, NK cells and monocytes. Three-colour staining revealed that the increase of ED9(+) monocytes was most likely due to the mobilization of two distinct monocyte subsets, predominantly ED9(+)CD4(-)NKR-P1A(+) and ED9(+)CD4(+)NKR-P1A(+). ELISA analysis revealed significantly elevated TNF-alpha levels in obese animals compared to their lean littermates, while IL-6 failed to show notable changes. In conclusion, the data of the present study revealed that leptin application induces a nutrition- and application-site dependent increase of circulating NK cells, granulocytes and specific monocyte subsets.