Growth factors in serum-free conditioned media from human bone marrow-derived mesenchymal stem cells (MSC-CM) are known to be effective in bone regeneration. However, the secretomes in MSC-CM that act as active ingredients for bone regeneration, as well as their mechanisms, remains unclear. Exosomes, components of MSC-CM, provide the recipient cells with genetic information and enhance the recipient cellular paracrine stimulation, which contributes to tissue regeneration. We hypothesized that MSC-CM-derived exosomes (MSC-Exo) promoted bone regeneration, and that angiogenesis was a key step. Here, we prepared an MSC-Exo group, MSC-CM group, and Exo-antiVEGF group (MSC-Exo with angiogenesis inhibitor), and examined the osteogenic and angiogenic potential in MSCs. Furthermore, we used a rat model of calvaria bone defect and implanted each sample to evaluate bone formation weekly, until week 4 after treatment. Results showed that MSC-Exo enhanced cellular migration and osteogenic and angiogenic gene expression in MSCs compared to that in other groups. In vivo, early bone formation by MSC-Exo was also confirmed. Two weeks after implantation, the newly formed bone area was 31.5 ± 6.5% in the MSC-Exo group while those in the control and Exo-antiVEGF groups were 15.4 ± 4.4% and 8.7 ± 1.1%, respectively. Four weeks after implantation, differences in the area between the MSC-Exo group and the Exo-antiVEGF or control groups were further broadened. Histologically, notable accumulation of osteoblast-like cells and vascular endothelial cells was observed in the MSC-Exo group; however, fewer cells were found in the Exo-antiVEGF and control groups.In conclusion, MSC-Exo promoted bone regeneration during early stages, as well as enhanced angiogenesis. Considering the tissue regeneration with transplanted cells and their secretomes, this study suggests that exosomes might play an important role, especially in angiogenesis.
Postischemic motor and somatosensory functions were significantly correlated with regional infarction volumes in the corresponding cortical regions. In gerbils, visual abnormality could be independently detected by the T-maze test. Such regional analyses of ischemic lesions would be useful for investigating the functional outcomes of stroke therapy.
The fate of postischemic tissues containing eosinophilic neurons (ENs), whether they remain viable or evolve into infarction, is largely unknown. We analyzed the time profile and distribution of ENs, reactive astrocytes (RAs), and infarction after transient cerebral ischemia. Unilateral forebrain ischemia was induced in Mongolian gerbils by two 10-min unilateral common carotid artery occlusions with a 5-h interval, and the brains at 24 h, 4 days, and 2, 4, and 16 weeks were prepared for morphometric analysis. Intra-ischemic laser Doppler flowmetry revealed significant ischemia, deeper in the anterior cortex, during carotid occlusion. Here, ENs appeared in the middle and deep layers at 24 h postischemia, and EN areas had extended to all cortical layers by 4 days. Large areas of high EN density turned into infarcts between 4 days and 4 weeks. In the posterior cortex, middle and deep cortical layers evolved low EN density areas without subsequent transformation into infarcts. RAs were consistently observed in areas with ENs, and RA areas with high EN density were largely transformed into infarcts between 4 days and 4 weeks postischemia. Areas of high, but not low, EN density were slowly transformed into infarcts after transient cerebral ischemia. Delayed astrocytic death took place in the RA areas with high EN density. In conclusion, density of ENs is an important indicator of delayed astrocytic death and infarction in postischemic tissue.
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