Exosomes from conditioned media of bone marrow-derived mesenchymal stem cells promote bone regeneration by enhancing angiogenesis

Abstract

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.

The Application of MSCs-Derived Extracellular Vesicles in Bone Disorders: Novel Cell-Free Therapeutic Strategy

“…MSCs-exosomes increased hMSC migration, induced mineral deposition and enhanced the differentiation potential of hMSCs into osteoblasts. In addition, MSCs-exosomes enhanced the expression of osteogenesis-related and angiogenesis-related genes, such as COL I, ALP, and VEGF, and promoted bone formation in vivo (Takeuchi et al, 2019). The CD9 −/− mouse, which is an established strain with low-exosome productivity, shows a significant delay in endochondral ossification and fracture healing compared with the wild-type mouse.…”

Extracellular vesicles therapy: opportunities, mechanisms and perspectives

“…В частности, имеются указания на стимулирующие регенерацию эффекты при ишемическом и ишемическом/реперфузионном повреждении клеток миокарда [2][3][4][5][6][7][8][9][10][11][12], на противоопухолевую эффективность внутриклеточных везикул [13][14][15][16][17], а также эффективность при терапии осложнений сахарного диабета [18][19][20]. Имеются данные о проостеогенном потенциале внутриклеточных везикул, что может найти применение при замещении костных дефектов [21][22][23][24]. Регенераторный потенциал рассматривается также и при повреждении печени [25][26][27], лёгких [28,29], нервной ткани [30][31][32][33][34][35], кожных покровов [36,37] и микроциркуляторного русла [38].…”

“…Интересно, что МСК отреагировали на введение ВВ уменьшением апоптоза за счёт увеличение экспрессии Bcl2 при активации каскада Wnt5a [28]. На МСК действие ВВ оказалось схожим: ангиогенный эффект обусловлен индукцией генов ангиогенеза (VEGF, ANG1, ANG2) и остеосинтеза (COL-I, ALP, OCN, OPN) [21], а пролиферативный -экспрессией ACAN, SOX-9, COL2A1 [53].…”

“…Доказано, что везикулярная микроРНК-196a способствовала индукции генов, ответственных за остеосинтез: ALP, OCN, OPN, Runx2 [24]. В других исследованиях установлено, что под действием везикул происходит усиление экспрессии остеогенных факторов -OCNи ангиогенных -VEGF [21,23], а также увеличиваются концентрации кальция и фосфора и фосфорилированных белков [23].…”

Umbilical Cord Mesenchymal Stem Cell-Derived Nanovesicles Potentiate the Bone-Formation Efficacy of Bone Morphogenetic Protein 2

“…Exosomes are extracellular vesicles ranging in size from to 40-200 nm and are secreted from most cell types [ 17 ]. Released exosomes are internalized in target cells and subsequently affect their biological behaviors and promote the tissue regenerative process [ 18 , 19 , 20 ]. Exosomes secreted from mesenchymal stem cells (MSCs) can promote the proliferation, migration, and tube formation of endothelial cells in vitro, angiogenesis in vivo [ 21 , 22 , 23 , 24 ], and in vivo bone regeneration [ 25 , 26 , 27 ].…”