Mesenchymal Stem Cells Derived Exosomes Promotes Bone Regeneration through Cystathionine‐β‐synthase Enhanced Angiogenesis and Osteogenesis during Bone Development

Abstract

It is critical that bone formation and angiogenesis are tightly coordinated during bone development and fracture healing, but the molecular regulator of such intercellular communication in bone microenvironment are not well studied. Cystathionine‐β‐synthase (CBS), an enzyme in homocysteine (Hcy) metabolic pathway play crucial role in osteoblast differentiation. Therefore, we hypothesize that CBS as a novel molecular regulator in osteoblasts, which regulates vascularization during bone development in osteoblast derived exosome. To test this hypothesis, we have used 7–10 weeks old male mice in our study; wild type mice (C57BL/6, WT) and CBS+/− mice. Blocking the CBS function was done by either inhibitor hydroxylamine (HA) or SiRNA prevented osteoblast differentiation and mineralization. This was supported by studies using osteoblasts cultured from bone marrow of CBS deficient (CBS+/−) mice. Exosomes of mesenchymal stem cell derived osteoblasts cultured stimulated endothelial migration and angiogenesis, which was prevented by blocking CBS in osteoblasts using HA or SiRNA. Mass spectrometry analysis and ELISA assay identified among others vascular endothelial growth factor (VEGF)‐A and VEGF homolog placental growth factor (PIGF) to be present in control osteoblasts culture medium and CBS(+/+) mice osteoblasts but not in osteoblasts treated with HA and CBS+/− osteoblasts. Immunostaining and metatarsal angiogenesis assay showed retarded vascularization in bone tissue of CBS+/− mice. However, osteopenia phenotype was confirmed in CBS+/− mice by microCT analysis. CBS+/− mice showed significantly reduced bone mineral density (BMD) and bone volume/tissue volume (BV/TV) and showed increased plasma Hcy levels compared to CBC+/+ mice (WT). However, CBS+/− mice exhibited a more significant increase in the number of TRAP‐positive osteoclasts and higher levels of RANKL than CBS+/+ mice. This suggests that Hcy levels may be associated with osteoclast activity. Furthermore, real‐time PCR and western blot analyses revealed significant decreases Alkaline phosphatase (ALP) and Runt‐related transcription factor 2 (RUNX2) expression in CBS‐deficient (CBS+/−) mouse bone marrow cells. In conclusion, we demonstrate that CBS in osteoblasts is at the crossroad of osteoblast differentiation/mineralization and angiogenesis. These findings uncover the previously undefined molecular understanding of CBS that promote angiogenesis and osteogenesis in bone development and fracture repair.Support or Funding InformationThis work is financial supported from National Institute of health grant AR‐067667 and HL‐107640‐NT are greatly acknowledged.