Paracrine Effects Of Immunomodulatory Activated Bone Marrow Mesenchymal Stem Cells On Osteoblast Function

Part I. The isolation, identification and paracrine tests of Bonemarrow mesenchymal stem cellsObjective: Bone marrow mesenchymal stem cells (BM-MSCs) are multipotentprogenitor cells that can potentially differentiate into a variety of cell types, includingosteoblasts, adipocytes, chondrocytes, and tenocytes. For the novel advancements inresearch, the paracrine effects and immunosuppressive ability of BM-MSC havegradually gained wide attention. In this research, BM-MSC were pretreated with bothIFN-γ and TNF-α, then we study its paracrine function changes by detecting the level ofbioactive factors in BM-MSCs conditioned medium (MSC-CM).Methods: we get BM-MSCs from normal fracture patients and identified by flowcytometry. BM-MSCs were differentiated into osteoblasts, adipogenic cells andchondroblasts in different inducer. BM-MSC were pretreated with both IFN-γ andTNF-α. The MSC-conditioned medium was prepared and analysed. Levels of TGF-β,HGF and VEGF were determined by enzyme-linked immunosorbent assay using ELISAcommercially available kits. IL-2, IL-4, IL-6and IL-10were determined by cytometricbead array (CBA).Results:We collect bone marrow from8normal fracture patients. All of that bone marrowwas cultured primary BM-MSCs. Primary cells arranged in a helical or spiralshape.This BM-MSCs expressed CD44, CD73and CD90without expression of CD34by flow cytometry detection.The3type of differentiation were explored by Alizarin Red staining, oil red Ostaining and Alcian blue staining, and we observed the specific substance formation. The secretion of BM-MSC are dramatically altered after activation by IFN-γ andTNF-α, and the secretions of IL-6, HGF, VEGF and TGF-β were significantly elevated(p<0.01). Furthermore, secretions of IL-2, IL-4and IL-10were unchanged afteractivation (p>0.05).Conclusions:The primary bone marrow mesenchymal stem cells were grow well and BM-MSCwere passaged genetic stability.BM-MSCs have strong differentiation characteristics, it can be used as an ideal cellin cell engineering.The paracrine effects of BM-MSC are dramatically altered after activation byIFN-γ and TNF-α, and the secretions of IL-6, HGF, VEGF and TGF-β weresignificantly elevated. Part II. Effects of immunomodulatory activated bone marrowmesenchymal stem cells on osteoblastObjective: Refractory nonunion and bone defects caused by high-energy damageshowed higher incidence, while there were still great difficulty in treatment. The lowernumber and activity of osteoblasts of fracture have great relation with nonunion. Ourprevious study showed the paracrine effects of BM-MSC are dramatically altered afteractivation by IFN-γ and TNF-α, and the secretions of IL-6, HGF, VEGF and TGF-βwere significantly elevated. In this research we explore the effects ofimmunomodulatory activated MSCs on osteogenesis by investigating the paracrineeffects of MSCs on osteoblasts in vitro.Methods: Proliferation of MG63was analysed by CCK8assay, and wound healingpotential of the cells was analysed by scratch assay. The MG63cytoskeleton wasobserved by rhodamine–phalloidin staining. Real-time PCR and immunofluorescencestaining were performed to evaluate osteoblast differentiation markers of ALP, COLI,OCN and OPN. Alizarin red staining was performed to evaluate osteoblastmineralization. Results: After treatment with conditioned medium collected from MSCs pretreatedwith cytokines, the proliferation and migration of MG63cells were significantlyimproved. The mRNA expression of osteoblast differentiation markers, ALP, COLI,OCN and OPN, was markedly higher after4–7d of induction (p<0.05). Furthermore,the immunofluorescence staining of MG63cells showed more OPN-positive cells andAlizarin red staining showed increased formation of calcium nodule after the IFN-γ andTNF-α combined pretreatment. No significant difference in cell morphology wasobserved.Conclusions: The paracrine effects of immunomodulatory activated MSCs cansignificantly promote osteoblast proliferation, migration, differentiation, mineralizationand ultimately enhance osteogenesis. The results of this study present a new directionfor the clinical application of MSCs in the repair of bone defects.