Assessment of tobacco heating system 2.4 on osteogenic differentiation of mesenchymal stem cells and primary human osteoblasts compared to conventional cigarettes

BACKGROUND Cigarette smoking(CS)is the most common method of consuming tobacco.Deleterious effects on bone integrity,increased incidence of fractures,and delayed fracture healing are all associated with CS.Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and,via the bloodstream,reach the skeletal system.New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems,such as e-cigarettes and tobacco heating systems(THS).THS are designed to heat tobacco instead of burning it,thereby reducing the levels of harmful toxic compounds released.AIM To examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS.METHODS Human immortalized mesenchymal stem cells(n=3)and primary human preosteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen(n=5)were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4“IQOS”or conventional“Marlboro”cigarettes for up to 21 d.Cell viability was analyzed using resazurin conversion assay(mitochondrial activity)and calcein-AM staining(esterase activity).Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase(AP)activity,while matrix formation was analyzed through alizarin red staining.Primary cilia structure was examined by acetylatedα-tubulin immunofluorescent staining.Free radical production was evaluated with 2’,7’-dichlorofluoresceindiacetate assay.RESULTS Our data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity(P<0.001).No significant differences in cytotoxicity between the diverse flavors of THS were observed.Harmful effects from THS on bone cell function were observed only at very high,non-physiological concentrations.In contrast,extracts from conventional cigarettes significantly reduced the AP activity(by two-fold)and matrix mineralization(four-fold)at low concentrations.Additionally,morphologic analysis of primary cilia revealed no significant changes in the length of the organelle involved in osteogenesis of osteoprogenitor cells,nor in the number of ciliated cells following THS treatment.Assessment of free radical production demonstrated that THS induced significantly less oxidative stress than conventional CS in osteoprogenitor cells.CONCLUSIONTHS was significantly less harmful to osteoprogenitor cells during osteogenesisthan conventional CS. Additional studies are required to confirm whether THS isa better alternative for smokers to improve delays in bone healing followingfracture.

Human adipose derived stem cells are superior to human osteoblasts (HOB) in bone tissue engineering on a collagen-fibroin-ELR blend

The ultrastructure of the bone provides a unique mechanical strength against compressive, torsional andtensional stresses. An elastin-like recombinamer (ELR) with a nucleation sequence for hydroxyapatitewas incorporated into films prepared from a collagen-silk fibroin blend carrying microchannel patternsto stimulate anisotropic osteogenesis. SEM and fluorescence microscopy showed the alignment ofadipose-derived stem cells (ADSCs) and the human osteoblasts (HOBs) on the ridges and in the groovesof microchannel patterned collagen-fibroin-ELR blend films. The Young's modulus and the ultimatetensile strength (UTS) of untreated films were 0.58 ± 0.13 MPa and 0.18 ± 0.05 MPa, respectively. After 28days of cell culture, ADSC seeded film had a Young's modulus of 1.21 ± 0.42 MPa and UTS of0.32 ± 0.15 MPa which were about 3 fold higher than HOB seeded films. The difference in Young'smodulus was statistically significant (p: 0.02). ADSCs attached, proliferated and mineralized better thanthe HOBs. In the light of these results, ADSCs served as a better cell source than HOBs for bone tissueengineering of collagen-fibroin-ELR based constructs used in this study. We have thus shown theenhancement in the tensile mechanical properties of the bone tissue engineered scaffolds by usingADSCs.

EFFECTS OF PARATHYROID HORMONE AND ESTRADIOL ON PROLIFERATION AND FUNCTION OF HUMAN OSTEOBLASTS FROM FETAL LONG BONE AN IN VITRO STUDY

The effect of parathyroid hormone (PTH) (0.01 nM-10 nM) and 17 -estradiol (E2, 1 nmol-10 nM) alone or in combination on 3H thymidine incorporation, alkaline phosphatase and adenylate cyclase activities were investigated in human fetal osteoblasts using serum-free monolayer primary cultures. The results showed that PTH inhibited cell proliferation while E, promoted it. On alkaline phosphatase activity, PTH showed a complex results while E, were slightly inhibitory. PHT-E2 combination suggested that E2 could alter the effect of PTH alone, also potentiated the anabolic and antagonize the catabolic effects of PTH on bone formation.

Algal oligosaccharides ameliorate osteoporosis via up-regulation of parathyroid hormone 1-84 and vascular endothelial growth factor

OBJECTIVE: To determine whether algal oligosaccharide affects the levels of parathyroid hormone 1-84(PTH1-84) and vascular endothelial growth factor(VEGF).METHODS: An osteoporosis rat model was established via bilateral ovariectomy. The model rats were fed algal oligosaccharides(molecular weights:600-1, 200 Da) for 4 months. Bone mineral density(BMD) was then measured. MG-63 human osteoblastic cells were treated with algal oligosaccharides. The expression of PTH1-84 and VEGF was then examined. Oligosaccharide-treated cells were transfected with PTH1-84 short hairpin RNA(sh RNA), VEGF sh RNA, and PTH1-84-VEGF small interfering RNA(si RNA). The growth rates were then compared between transfected and non-transfected cells.RESULTS: Algal oligosaccharides increased the BMD of the osteoporosis rat model compared with untreated controls(P < 0.05). When MG-63 cells were treated with algal oligosaccharides, the growth rate increased by 25% compared with the control group at day 3(P < 0.05). In addition, the expression of PTH84 and VEGF was enhanced. Con-versely, when the cells were transfected with PTH84 sh RNA, VEGF sh RNA, or PTH1-84-VEGF si RNA, the growth rate was decreased by 17%, 35% and 70%, respectively, compared with controls at day 3(P < 0.05).CONCLUSION: Algal oligosaccharides ameliorate osteoporosis via up-regulation of PTH1-84 and VEGF. Algal oligosaccharides should be developed as a potential drug for osteoporosis treatment.