Applications of nanoparticles (NP) in medicine, industry and other branches of human activities undoubtedly contribute to technology development and well-being. However, as NP are very small units in a wide range of materials, there is a lack of information related to possible side effects potentially affecting the health of organisms. There is increasing experimental interest in the determination of environmental effects on humans exposed to NP. Most such experimental studies focus on adult populations or adult experimental animals. However, embryos can be more sensitive to pollutants and environmental impacts in some species. Therefore, some investigations dealing particularly with the effects of NP on embryonic development have appeared recently and this issue is becoming of great concern. The aim of this review is to summarize the knowledge on the effects of various nanomaterials on embryonic development. A comprehensive collection of significant experimental nanotoxicity data is presented, which also indicate how the toxic effect of NP can be mediated and modulated with respect to possible effective protection strategies.

Caspases are proteases traditionally associated with inflammation and cell death. Recently, they have also been shown to modulate cell proliferation and differentiation. The aim of the current research was to search for osteogenic molecules affected by caspase inhibition and to specify the individual caspases critical for these effects with a focus on proapoptotic caspases: caspase-2,-3,-6,-7,-8 and-9. Along with osteocalcin (Ocn), general caspase inhibition significantly decreased the expression of the Phex gene in differentiated MC3T3-E1 cells. The inhibition of individual caspases indicated that caspase-8 is a major contributor to the modification of Ocn and Phex expression. Caspase-2 and-6 had effects on Ocn and caspase-6 had an effect on Phex. These data confirm and expand the current knowledge about the nonapoptotic roles of caspases and the effect of their pharmacological inhibition on the osteogenic potential of osteoblastic cells. Caspases are proteases that are currently associated with inflammation and cell death. Their use has broad implications for pathological conditions, such as cancer and degenerative disorders. Caspase inhibitors have been tested in several therapeutic approaches 1. Additionally, a much broader spectrum of caspase functions has been demonstrated 2 , particularly proapoptotic caspases, including apical caspases-8 and-9, the executive trio of caspase-3,-6 and-7 and the still enigmatic caspase-2. New functions of caspases have also been reported in osteogenesis 3,4. Bmp4-induced differentiation of osteoblastic MC3T3-E1 cells leads to the activation of caspase-2,-3 and-8 without increasing the apoptosis rate 5. Pharmaceutical inhibition of caspases reduced Alp activity in MC3T3-E1 cells and the expression levels of osteocalcin, a molecule typically found in osteoblasts 4,5. Notably, osteocalcin is also used in medical diagnoses as a biochemical marker of bone formation and metabolic risk 6. Pharmacological caspase inhibitors are considered potential tools in several therapies 1. Previous works have focused on the alteration of gene expression during MC3T3-E1 cell differentiation 7,8 , which consists of several phases, including proliferation, differentiation and matrix deposition, accompanied by the production of specific osteogenic factors 9. Since these phases were first characterized 10 , hundreds of reports have been based on experiments performed using MC3T3-E1 cells. Given their intramembranous origin, MC3T3-E1 cells are recognized as suitable in vitro models for direct osteoblastic differentiation and pleiotropic studies 11. Based on published results and our preliminary data, we hypothesized that proapoptotic caspases impact gene expression in differentiated MC3T3-E1 cells. The following investigation addresses the effects of caspase blockade on gene expression in differentiated cells. For the first time, a cell bioluminescence-based approach was used to record the activation of individual proapoptotic caspases during MC3T3-E1 cell cultivation. Therefore, the osteogenic profi...

Caspases are well known proteases in the context of inflammation and apoptosis. Recently, novel roles of pro-apoptotic caspases have been reported, including findings related to the development of hard tissues. To further investigate these emerging functions of pro-apoptotic caspases, the in vivo localisation of key pro-apoptotic caspases (-3,-6,-7,-8, and -9) was assessed, concentrating on the development of two neighbouring hard tissues, cells participating in odontogenesis (represented by the first mouse molar) and intramembranous osteogenesis (mandibular/alveolar bone). The expression of the different caspases within the developing tissues was correlated with the apoptotic status of the cells, to produce a picture of whether different caspases have potentially distinct, or overlapping non-apoptotic functions. The in vivo investigation was additionally supported by examination of caspases in an osteoblast-like cell line in vitro. Caspases-3,-7, and -9 were activated in apoptotic cells of the primary enamel knot of the first molar; however, caspase-7 and -8 activation was also associated with the non-apoptotic enamel epithelium at the same stage and later with differentiating/differentiated odontoblasts and ameloblasts. In the adjacent bone, active caspases-7 and -8 were present abundantly in the prenatal period, while the appearance of caspases-3,-6, and -9 was marginal. Perinatally, caspases-3 and -7 were evident in some osteoclasts and osteoblastic cells, and caspase-8 was abundant mostly in osteoclasts. In addition, postnatal activation of caspases-7 and -8 was retained in osteocytes. The results provide a comprehensive temporo-spatial pattern of pro-apoptotic caspase activation, and demonstrate both unique and overlapping activation in non-apoptotic cells during development of the molar tooth and mandibular/alveolar bone. The importance of caspases in osteogenic pathways is highlighted by caspase inhibition in osteoblast-like cells, which led to a significant decrease in osteocalcin expression, supporting a role in hard tissue cell differentiation.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.