BackgroundFor an effective bone graft for reconstruction of the maxillofacial region, an adequate vascular network will be required to supply blood, osteoprogenitor cells, and growth factors. We previously reported that the secretomes of bone marrow-derived mesenchymal stem cells (MSC-CM) contain numerous growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-β1, and vascular endothelial growth factor (VEGF), which can affect the cellular characteristics and behavior of regenerating bone cells. We hypothesized that angiogenesis is an important step for bone regeneration, and VEGF is one of the crucial factors in MSC-CM that would enhance its osteogenic potential. In the present study, we focused on VEGF in MSC-CM and evaluated the angiogenic and osteogenic potentials of MSC-CM for bone regeneration.MethodsCytokines in MSC-CM were measured by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were cultured with MSC-CM or MSC-CM with anti-VEGF antibody (MSC-CM + anti-VEGF) for neutralization, and tube formation was evaluated. For the evaluation of bone and blood vessel formation with micro-computed tomography (micro-CT) and for the histological and immunohistochemical analyses, a rat calvarial bone defect model was used.ResultsThe concentrations of IGF-1, VEGF, and TGF-β1 in MSC-CM were 1515.6 ± 211.8 pg/mL, 465.8 ± 108.8 pg/mL, and 339.8 ± 14.4 pg/mL, respectively. Tube formation of HUVECs, bone formation, and blood vessel formation were increased in the MSC-CM group but decreased in the MSC-CM + anti-VEGF group. Histological findings suggested that new bone formation in the entire defect was observed in the MSC-CM group although it was decreased in the MSC-CM + anti-VEGF group. Immunohistochemistry indicated that angiogenesis and migration of endogenous stem cells were much more abundant in the MSC-CM group than in the MSC-CM + anti-VEGF group.ConclusionsVEGF is considered a crucial factor in MSC-CM, and MSC-CM is proposed to be an adequate therapeutic agent for bone regeneration with angiogenesis.

Peripheral nerve regeneration across nerve gaps is often suboptimal, with poor functional recovery. Stem cell transplantation-based regenerative therapy is a promising approach for axon regeneration and functional recovery of peripheral nerve injury; however, the mechanisms remain controversial and unclear. Recent studies suggest that transplanted stem cells promote tissue regeneration through a paracrine mechanism. We investigated the effects of conditioned media derived from stem cells from human exfoliated deciduous teeth (SHED-CM) on peripheral nerve regeneration. In vitro, SHED-CM-treated Schwann cells exhibited significantly increased proliferation, migration, and the expression of neuron-, extracellular matrix (ECM)-, and angiogenesis-related genes. SHED-CM stimulated neuritogenesis of dorsal root ganglia and increased cell viability. Similarly, SHED-CM enhanced tube formation in an angiogenesis assay. In vivo, a 10-mm rat sciatic nerve gap model was bridged by silicon conduits containing SHED-CM or serum-free Dulbecco's modified Eagle's medium. Light and electron microscopy confirmed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in the SHED-CM group at 12 weeks after nerve transection surgery. The sciatic functional index (SFI) and gastrocnemius (target muscle) wet weight ratio demonstrated functional recovery. Increased compound muscle action potentials and increased SFI in the SHED-CM group suggested sciatic nerve reinnervation of the target muscle and improved functional recovery. We also observed reduced muscle atrophy in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. SHED-CM may therefore provide a novel therapy that creates a more desirable extracellular microenvironment for peripheral nerve regeneration.

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Background : Mesenchymal stem cells (MSCs) secrete various kinds of soluble factors such as exosomes. Exosomes are nanoparticles and are thought to play essential roles in intercellular communication. We collected exosomes from the conditioned media from human MSCs (MSCs-Exo) and reported that MSCs-Exo enhanced bone formation. Recent studies reported that harsh environments such as a low oxygen environment stimulate cells and change the composition of their soluble factors.Aim/Hypothesis : The aim of this study is to evaluate the bone regenerative efficacy of MSCs-Exo stimulated by hypoxia.Materials and Methods : hMSCs were purchased from LONZA. They were cultured in Dulbecco ' s modifi ed Eagle ' s medium with exosome-free serum at 1% O2 using the BIONIX hypoxic culture kit (Sugiyamagen, Tokyo, Japan), and then the exosomes (hypo-MSCs-Exo) were collected using an ultra-centrifuge method. They were observed using transmission electron microscopy and their particle size distribution was evaluated by Nanoparticle tracking analysis. Profiling of microRNAs contained in hypo-MSCs-Exo was performed by microarray.Rat mesenchymal stem cells (rMSCs) and human umbilical endothelial cells (HUVECs) were cultured with hypo-MSCs-Exo or MSCs-Exo for 48 hours. Then, alizarin red S staining, real-time RT-PCR analysis, and tube formation assay were performed. Rat calvarial bone defect models (5 mm in diameter) were made, and then hypo-MSCs-Exo or MSCs-Exo were applied into the defects with atelocollagen sponges. After 4 weeks, the new bone formation was evaluated by radiographical and histological analyses.Results : hypo-MSCs-Exo or MSC-Exos were observed as round-shaped 70-200 nm nanoparticles. Profiling of microRNAs revealed that there were many kinds of miRNAs contained in hypo-MSCs-Exo and they were the different composition from those contained in MSCs-Exo.Compared with MSCs-Exo, when hypo-MSCs-Exo were applied into rMSCs, they have enhanced the osteogenic differentiation and the gene expression of Alkaline Phosphatase, Runt-related transcription factor 2, Collagen type 1 α 2. hypo-MSCs-Exo also enhanced tube formation of HUVECs. In vivo, new bone formation rate was significantly higher in the hypo-MSCs-Exo group compared with in the MSCs-Exo group. Conclusion and Clinical Implications: A low oxygen stimulated hMSCs and changed the composition of miRNAs contained in their exosomes and hypo-MSCs-Exo enhanced osteogenesis and angiogenesis.