Introduction Tobacco smoking has been implicated in an array of adverse health outcomes, including those that affect adult bone. However, little is known about the impact of tobacco products on developing bone tissue as it develops in the embryo. Methods Here, human embryonic stem cells were differentiated into osteoblasts in vitro and concomitantly exposed to various concentrations of smoke solutions from two conventional, one additive-free and two harm reduction brands of cigarettes. Differentiation inhibition was determined by calcium assays that quantified matrix mineralization and compared to the cytotoxicity of the tobacco product. Results Exposure to mainstream smoke from conventional and additive-free cigarettes caused no inhibition of cell viability or mineralization, while sidestream smoke concentration-dependently produced cell death. In contrast, mineralization was inhibited only by the highest mainstream concentration of harm-reduction smoke solution. Additionally, sidestream smoke solution from the harm-reduction cigarettes impeded calcification at concentrations lower than those determined to be cytotoxic for conventional products. Conclusions Sidestream smoke impaired in vitro osteogenesis at sub-toxic concentrations. In addition, though often perceived as safer, smoke from harm reduction cigarettes was more potent in inhibiting in vitro osteogenesis than smoke from conventional cigarettes. Implications This study adds to a growing list of adverse outcomes associated with prenatal tobacco exposure. Specifically, in vitro exposure to tobacco products interfered with osteogenic differentiation of human embryonic stem cells, a well-established surrogate model for human embryonic bone development. Contrasting a diverse array of tobacco products unveiled that sidestream smoke was generally more developmentally osteotoxic than mainstream smoke and that harm-reduction products may not be less harmful than conventional products, adverse effects that were seemingly independent of nicotine.

Epidemiological studies suggest tobacco consumption as a probable environmental factor for a variety of congenital anomalies, including low bone mass and increased fracture risk. Despite intensive public health initiatives to publicize the detrimental effects of tobacco use during pregnancy, approximately 10-20% of women in the United States still consume tobacco during pregnancy, some opting for so-called harm-reduction tobacco. These include Snus, a type of orally-consumed yet spit-free chewing tobacco, which is purported to expose users to fewer harmful chemicals. Concerns remain from a developmental health perspective since Snus has not reduced overall health risk to consumers and virtually nothing is known about whether skeletal problems from intrauterine exposure arise in the embryo.

Traditional methods of quantifying osteoblast calcification in culture require the use of calcium sensitive dyes, such as Arsenazo III or Alizarin Red S, which have been successfully used for decades to assess osteogenesis. Because these dyes elicit a colorimetric change when reacted with a cell lysate and are cytotoxic to live cells, they forfeit the ability to trace calcification longitudinally over time. Here, we demonstrate that image analysis and quantification of calcification can be performed from a series of time-lapse images acquired from videos. This method capitalizes on the unique facet of the mineralized extracellular matrix to appear black when viewed with phase contrast optics. This appearance of calcified areas had been previously documented to be characteristic to the formation of bone nodules in vitro. Due to this distinguishable appearance, extracting the information corresponding to calcification through segmentation allowed us to threshold only the pixels that comprise the mineralized areas in the image. Ultimately, this method can be used to quantify calcification yield, rates and kinetics facilitating the analyses of bone-supportive properties of growth factors and morphogens as well as of adverse effects elicited by toxicants. It may also be used on images that were acquired manually. The method is less error-prone than absorption-based assays since it takes longitudinal measurements from the same cultures It is cost effective as it foregoes the use of calcium-sensitive dyes It is automatable and amenable to high-throughput and thus allows the concurrent quantification of multiple parameters of differentiation