Studies On The Effect Of Cerium On The Osteogenesis Of Bone Marrow Mesenchymal Stem Cells

Objective:Rare earths include lanthanide elements of the chemical periodic table as well as two other chemical elements scandium (Sc) and yttrium (Y) with similar chemical properties. Our country has the most abundant rare earth resources in the world; the production and reserves account for the first in the world. Cerium is a type of lanthanide; it has various functions, such as anti-bacterial, anti-emetic and anti-cancer functions. Since the rare earth ion Ce3+has similar ionic radius to calcium ions and higher charge than calcium ions, it has more affinity to intracellular calcium-binding sites and subsequently affect the bone cell functions and intervene in the bone remodeling process. There are reports on the effects of cerium ion on osteoblasts as early as in the20th century, the studies found the rare earth cerium ion (Ce3+) can promote the proliferation, differentiation and osteogenesis of osteoblast and the role of rare earth ions in regulating osteoblast cells depends on the two-way property of rare earth concentrations, the types of rare earth elements and cell properties.Mandible is a major scaffold bone of oral and maxillofacial region, exercising the functions of chewing, swallowing and language. Inflammation, trauma, tumor surgery and other reasons can cause mandibular defects, reduce the quality of people’s lives. Physiological and functional reconstruction of the damaged tissues is many scholars’research focus. In recent years, the development of bone tissue engineering provides a new way for bone defect repair. Being easy to get and establish stable cell lines, bone marrow mesenchymal stem cells (BMSCs) are a class of seed cells for bone tissue engineering, which have a strong proliferation, multi-differentiation potential and simple and mature in vitro culture process. BMSCs can differentiate into osteoblasts under in vitro-and in vzvo-induced environment. In the process of BMSCs directionally transforming into osteoblasts, the osteoblasts express specific markers of bone formation, such as ALP, Runx2, OCN, Satb2, BSP, etc.; in the late stage of osteoblast differentiation, cells begin to mineralize, intracellular ALP decreases, hydroxyapatite crystals are formed in the extracellular matrix secreted by osteocalcins and other non-collagenous proteins, finally calcium deposits to form a calcium nodules. ALP, Runx2, Satb2are early gene markers for osteoblasts, and OCN is marker gene for late differentiation.Bone defect repair is a complex process; many factors affect cell proliferation, differentiation and bone matrix formation in the process of bone repair; in-depth study of bone morphogenetic proteins (BMPs) was conducted in the area of new bone formation. As a class of bone-inducing factor, BMP2is the most representative one in BMPs family, which can promote cell proliferation at the site of bone injury, increase ALP activity and subsequently promote BMSCs to differentiate into osteoblasts. Extracellular BMP2binds with BMPR II on the cell membrane and phosphorylates BMPR II to activate BMPR I; the activated BMPR I phosphorylates intracellular Smadl/5/8to form a complex, then the complex enters into the nucleus, bind to the DNA sequence, thereby regulate the transcription of BMP target genes.In conclusion, the effect of Ce3+on BMSCs osteogenesis has been rarely reported, although there are already reports on Ce3+promoting the proliferation and osteogenesis of osteoblasts. BMSCs osteogenesis can be achieved because of their multi-differentiation potential, however, the mechanisms of BMSCs osteogenesis had not been disclosed due to the limitation of methods and the complexity of cell signaling pathways as well as the cytokine regulating network characteristics. In this study, the effect of rare earth cerium(III) ion on BMSCs osteogenesis was conducted by in vivo and in vitro experiments, and the molecular mechanisms of BMSCs osteogenesis promoted by Ce3+were investigated preliminarily, which provides a new theoretical basis for BMSCs osteogenesis and new ideas for the clinical treatment of bone defects.Materials and methods:1. Mice BMSCs culture, identification and effect of Ce3+on BMSCs proliferation, osteogenesis and related gene expressions.1) Isolation, culture and identification of BMSCs from miceBMSCs was isolated and cultured from C57BL/6mice by bone marrow adherent method. Then BMSCs was identified through the following experiments:medium changing and limited dilution process to purify BMSCs; MTT assay to plot cell growth curve; flow cytometry to test BMSCs surface antigens; BMSCs osteogenesis and adipogenic differentiation.2) Effect of cerium(Ⅲ) ion on BMSCs proliferation, osteogenesis and related gene expressions.When BMSCs were in logarithmic growth phase, the effect of cerium(Ⅲ) ion on the BMSCs proliferation was examined by CCK-8assay. ALP activity test and "calcium nodules" Alizarin Red S staining was used to determine the effect of rare earth cerium (Ⅲ) ion on BMSCs osteogenesis at the early and late stage. Use qRT-PCR and Western blotting to identify the expressions of osteoblast genes Runx2, Satb2, OCN mRNA and related proteins after cultured intervention by cerium(Ⅲ) ion.2. The impact of Ce3+on BMSCs migration and molecular mechanisms of BMSCs migration and osteogenesis promoted by Ce3+.1) The effect of cerium (Ⅲ) ion on BMSCs migration.Use Transwell chamber to detect the effect of Ce3+on BMSCs migration; it is well known that SDF-1is the main chemokine for stem cell migration and CXCR4is SDF-1natural receptor; so qRT-PCR was used to detect SDF-1and CXCR4gene expressions after7-day cerium(Ⅲ) ion intervened culture.2) Molecular mechanisms of BMSCs migration and osteogenesis promoted by Ce3+.Firstly, the BMP2mRNA expression of BMSCs was detected by qRT-PCR assay. Secondly, we use western blot to detect phosphorylated Smad1/5/8(p-Smadl/5/8) protein expressions of BMSCs treated with Ce3+for0,10,20,30min, respectively; we also examined the subcellular localization of p-Smadl/5/8through immunofluorescence experiments. Furthermore, BMSCs were pretreated with or without LDN-193189(0.5μM), which is an inhibitor for BMP type Ⅰ receptor; then the treated BMSCs were incubated with or without0.001μM Ce3+; lastly, p-Smad 1/5/8protein expressions were examined by western blot assay, and BMP2, SDF-1and Runx2gene expressions were then detected by qRT-PCR technology.3. Experimental study of skull defect repair in mice by BMSCs cell sheet after cerium(III) ion intervention culture.Bone defects were created in the skull of7-week-old male C57BL/6mice and these mice were randomly divided into three groups without damaging their duras, of which six were experimental group implanted by BMSCs+Ce3+cell sheet, six were control group implanted by BMSCs cell sheet, six were blank control group without any implantation. The animals were sacrificed after4weeks and specimens were reserved and made into tissue sections. After hematoxylin and eosin (HE) staining, histomorphometric analysis was performed to evaluate bone regeneration; the rate of new bone formation was calculated and the healing status of bone defects was observed. Finally, the effects of rare earth cerium(III) ion on osteoblast genes BSP, OCN expressions were investigated by specimen qRT-PCR.Results:1. Mice BMSCs culture, identification and the effect of Ce3+on BMSCs proliferation, osteogenesis and related gene expressions.1) Isolation, culture and identification of BMSCs from micePrimary BMSCs with stable morphology were obtained by whole bone marrow adherence method. Then the cells were purified by replacing the medium and limiting dilution. BMSCs growth curve was plotted "S" shape by MTT assay, showing that the adherent cells were at an initial rare density in the first2days, the cells replicated rapidly and reached almost80%confluence from3rd to7th days and the growth began to decrease in7-8days. Four known phenotypes were characterized to be associated with BMSCs by flow cytometric analysis. BMSCs were positive for CD44and CD29, and were negative for hematopoietic lineage markers CD34and CD45. After osteogenic and adipogenic differentiation induction, mineralized nodules and accumulated intracellular lipid droplets were detected with alizarin red staining and red0staining, respectively. Based on the above experimental results, we determined that the isolated and cultured cells were BMSCs, and BMSCs have multiple differentiation potentials. 2) Effect of cerium(Ⅲ) ion on BMSCs proliferation, osteogenesis and related gene expressions.The CCK-8assay and ALP activity test results revealed that Ce3+displayed a positive effect on proliferation and ALP activity of BMSCs at lower concentrations (0.001μM). It was also observed that there were more small round alizarin red-positive nodules formed in the BMSCs cultures on the21th day of induction with Ce3+(0.001μM) than without Ce3+. qRT-PCR assay and Western blot analysis results indicated that the expressions of Runx2, Satb2, OCN mRNA and related proteins were significantly up-regulated in the BMSCs treated with Ce3+(0.001μM) for7days as compared to control group.2. The effect of Ce3+on BMSCs migration and molecular mechanisms of osteogenic differentiation.1) Effect of cerium (Ⅲ) ion on BMSCs migration.Ce3+promotes BMSCs migration by promoting migration chemokine SDF-1gene expression, but no significant effect on the expression of CXCR4gene.2) Molecular mechanisms of BMSCs migration and osteogenesis promoted by Ce3+.First, it was found that Ce3+could increase the mRNA expression of BMP2in BMSCs and Ce3+could cause a time-dependent increase of p-Smad1/5/8protein expressions. Then it was also detected that p-Smadl/5/8located in the cytoplasm in the untreated BMSCs, whereas BMSCs treated with Ce3+for30minutes led to the preferential nuclear localization of p-Smadl/5/8. Finally, western blot analysis indicated that p-Smadl/5/8protein expressions were decreased in LDN-193189pretreated BMSCs. Whereas Ce3+treatment of BMSCs increased BMP2, SDF-1and Runx2mRNA expression, pre-treatment with LDN-193189(0.5μM) significantly blocked the Ce3+-mediated up-regulation of SDF-1and Runx2mRNA expression, but not influence the expression of BMP2mRNA.3. Experimental study of skull defect repair in mice by BMSCs cell sheet after cerium (Ⅲ) ion intervention culture.Through animal model experiment, Ce3+can promote new bone formation, and up-regulates osteogenic genes BSP, OCN. Conclusions:1. It is simple and easy to master BMSCs isolation and culture through whole bone marrow adherence method. Owning to their multi-differentiation potentials, BMSCs is an ideal seed cells for bone tissue engineering.2. Rare earth ion Ce3+can promote BMSCs proliferation, osteogenesis and recruitment.3. Rare earth ion Ce3+promotes BMSCs migration and osteogenic differation by BMP/Smad signaling pathways.4. Rare earth ion Ce3+can promote bone formation in vivo.