Carbon Black Inhibits The Osteogenic Differentiation Of Mesenchymal Stem Cells: The Role Of Mitochondria

Background: Carbon black(CB)is a fine black powder of nearly pure elemental carbon.The manufactured product of CB(Printex 90)has been used as a representative carbonaceous particle toxicant in air pollution studies.Potential health effects of nano-sized CB particles have been studied in vitro and in vivo.CB is responsible for a number of disorders,including early pulmonary response,vascular effects,genotoxicity,and reproductive toxicity.However,its effects on bone health remain unclear.Since exposure to elevated levels of CB is a growing problem in many regions of the world,an understanding of the effect of CB on bone health would be useful.Bone is a dynamic tissue with constant remodeling.During bone remodeling,bone marrow mesenchymal stem cells(MSCs)carry on constant differentiation and bone formation.MSCs are multipotent stem cells with the potential for self-renewal and differentiation.MSCs can be isolated from many tissues and have the capacity to differentiate into multiple cell lineages in vitro,including osteoblasts.The differentiation of MSCs requires increased energy,and mitochondria play an important role in the differentiation process.Healthy mitochondria are maintained by mitochondrial biogenesis as well as dynamic fusion and fission events,while damaged mitochondria are segregated and removed through fission and mitophagy.Furthermore,mitochondria are potential targets of nanomaterials,so we studied the role of mitochondria in the CB Printex 90-induced effects on osteogenesis.Results: 1.Under TEM Printex 90 was spherically shaped and the primary particle size was 14 nm.Printex 90 dispersed as small aggregates in culture medium with10% FBS.The isolated cells were positive for mesenchymal-associated markers,such as CD29,CD44,and CD90,with only a small percent of CD45 positive hematopoietic cells.Alizarin red S staining and oil red O staining confirmed thatprimary MSCs have a high potential ability to differentiate.2.TEM was used to find the presence of Printex 90 in MSCs confirming the uptake of Printex 90 by MSCs.Cell viability was significantly decreased with Printex 90 dosage and the decrease was dose-dependent.A Printex 90 dosage of 0.03 μg/ml or below had no obvious detrimental effect on cell viability.The cell viability was not affected after incubation with 3 ng/mL or 30 ng/ml Printex 90 in osteo-induction media for 7 d.Low doses of Printex 90(3 ng/ml and 30 ng/ml)significantly inhibited osteogenesis of MSCs.Printex 90 caused down-regμlation of osteoblastic markers,ALP,Bglap and Runx2;reduced activity of alkaline phosphatase(ALP);poor mineralization of osteogenically induced MSCs.3.Followed the extended exposure time of Printex 90,the gradual morphological changes of mitochondria appeared.Dissolved mitochondrial cristae,swelling and abnormal density of mitochondria in cultured MSCs were observed at 7 d exposure to30 ng/ml Printex 90.Under Printex 90,cellμlar ATP production was decreased,mitochondrial respiration(basal OCR,ATP-linked respiration,the maximal OCR,the spare respiratory capacity,non-mitochondrial respiration)was impaired with reduced expression of ATPase,and the mitochondrial membrane was depolarized.The quantity and quality of mitochondria are tightly controlled by mitochondrial biogenesis,mitochondrial dynamics and mitophagy.The contents of mitochondrial DNA(mtDNA)were decreased by Printex 90 treatment.The transcriptional co-activator and transcription factors for mitochondrial biogenesis,PGC-1α,Nrf1 and TFAM,were suppressed by Printex 90 treatment,suggesting that decreased biogenesis was caused by Printex 90 treatment during osteogenesis.The decreased fusion and fission related factors,Mfn1,Mfn2,Opa1,and Drp1,Fis1 demonstrated that mitochondrial fusion and fission were significantly inhibited by Printex 90 treatment.PINK1 accumμlated inPrintex 90-treated cells,and more Parkin wasrecruited to mitochondria,indicating that mitophagy increased to remove the damaged mitochondria.Conclusions: This is the first report of the inhibitory effects of CB on the osteogenesis of MSCs and the involvement of mitochondria in CB Printex 90-induced suppression of MSCs osteogenesis.Our findings have public health implications for developing regulatory strategies to reduce Printex 90 exposure and mitigate its harmful effects.