Prenatal exposure to environmental factors and congenital limb defects

Kobolak J., Dinnyes A., Memic A., Khademhosseini A., Mobasheri A.

Mesenchymal stem cells (MSCs) are multipotent stem cells. Although they were originally identified in bone marrow and described as 'marrow stromal cells', they have since been identified in many other anatomical locations in the body. MSCs can be isolated from bone marrow, adipose tissue, umbilical cord and other tissues but the richest tissue source of MSCs is fat. Since they are adherent to plastic, they may be expanded in vitro. MSCs have a distinct morphology and express a specific set of CD (cluster of differentiation) molecules. The phenotypic pattern for the identification of MSCs cells requires expression of CD73, CD90, and CD105 and lack of CD34, CD45, and HLA-DR antigens. Under appropriate micro-environmental conditions MSCs can proliferate and give rise to other cell types. Therefore, they are ideally suited for the treatment of systemic inflammatory and autoimmune conditions. They have also been implicated as key players in regenerating injured tissue following injury and trauma. MSC populations isolated from adipose tissue may also contain regulatory T (Treg) cells, which have the capacity for modulating the immune system. The immunoregulatory and regenerative properties of MSCs make them ideal for use as therapeutic agents in vivo. In this paper we review the literature on the identification, phenotypic characterization and biological properties of MSCs and discuss their potential for applications in cell therapy and regenerative medicine. We also discuss strategies for biomaterial micro-engineering of the stem cell niche.

Howard M., Tuan R.S., Wallis G.A.

Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skeleton. Both GDF5 and ERG are proposed as regulators of chondrocyte differentiation during and post interzone formation. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Erg expression was identified in mouse knee joints at E13.5. Expression analyses were performed using micromass cultures of murine C3H10T1/2 mesenchymal cells undergoing induced chondrogenesis in the presence and absence of GDF5 and ERG. At E13.5, Erg expression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expression profile was altered by the addition of recombinant GDF5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression identified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by GDF5. Erg regulation by GDF5 in induced mesenchymal cells in vitro is dependent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg expression causes downregulation of Col2a1 and Col10a1 expression and this effect is potentially mediated by Osr2 and/or Ube2b. Combined, these data suggest a possible pathway linking GDF5, ERG and downstream factors in the processes of chondrocyte differentiation during articular joint formation.

Tickle C.

The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors (FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein (BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs, respectively, the apical ectodermal ridge producing fibroblast growth factors and the polarizing region, Sonic hedgehog (Shh). These signals are the same in both wings/forelimbs and legs/hindlimbs and control growth and pattern formation by providing the mesoderm cells of the limb bud as it develops with positional information. The precise anatomy of the limb depends on the mesoderm cells in the developing bud interpreting positional information according to their identity - determined by Pitx1 in hindlimbs - and genotype. The competence to form a limb extends along the entire antero-posterior axis of the trunk - with Hox gene activity inhibiting the formation of forelimbs in the interlimb region - and also along the dorso-ventral axis.

Paradis F., Hales B.F.

Bedard T., Lowry R.B., Sibbald B., Kiefer G.N., Metcalfe A.

The birth prevalence of limb deficiencies in Alberta has been fluctuating. The objectives were to examine patterns and temporal trends of congenital limb deficiencies in Alberta and compare rates with those of other jurisdictions. The Alberta Congenital Anomalies Surveillance System data on live births, stillbirths, and terminations of pregnancy (

Vargesson N.

Nearly 60 years ago thalidomide was prescribed to treat morning sickness in pregnant women. What followed was the biggest man-made medical disaster ever, where over 10,000 children were born with a range of severe and debilitating malformations. Despite this, the drug is now used successfully to treat a range of adult conditions, including multiple myeloma and complications of leprosy. Tragically, a new generation of thalidomide damaged children has been identified in Brazil. Yet, how thalidomide caused its devastating effects in the forming embryo remains unclear. However, studies in the past few years have greatly enhanced our understanding of the molecular mechanisms the drug. This review will look at the history of the drug, and the range and type of damage the drug caused, and outline the mechanisms of action the drug uses including recent molecular advances and new findings. Some of the remaining challenges facing thalidomide biologists are also discussed.

Suter-Dick L., Alves P.M., Blaauboer B.J., Bremm K., Brito C., Coecke S., Flick B., Fowler P., Hescheler J., Ingelman-Sundberg M., Jennings P., Kelm J.M., Manou I., Mistry P., Moretto A., et. al.

Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.

Bradley E.W., Carpio L.R., Olson E.N., Westendorf J.J.

Goldfarb C.A., Wall L.B., Bohn D.C., Moen P., Van Heest A.E.

To examine the relative presentation frequency of children with upper limb congenital anomalies at 3 Midwestern referral centers using the Oberg, Manske, and Tonkin (OMT) classification and to assess the utility of this new classification system.641 individuals with 653 congenital upper extremity anomalies were identified at 3 hospitals in 2 large metropolitan areas during a 1-year interval. Patients were identified prospectively and the specific upper extremity anomaly and any associated syndromes were confirmed using medical records and radiographs. We applied the OMT classification that categorizes anomalies using a dysmorphology outline as malformations, dysplasias, deformations, and syndromes, and assessed its utility and ease of use.There were 480 extremities (74%) with a limb malformation including 184 involving the entire limb. Arthrogryposis was the most common of these (53 extremities). Anomalies affecting only the hand plate accounted for 62% (296) of the malformations. Of these, radial polydactyly (15%) was the most common specific anomaly, followed by symbrachydactyly (13%) and cleft hand (11%). Dysplasias were noted in 86 extremities; 55 of these were multiple hereditary exostoses. There were 87 extremities with deformations and 58 of these were trigger digits. A total of 109 children had a syndrome or association. Constriction ring sequence was most common. The OMT was straightforward to use and most anomalies could be easily assigned. There were a few conditions, such as Madelung deformity and symbrachydactyly, that would benefit from clarification on how to best classify them.Malformations were the most common congenital anomalies in the 653 upper extremities evaluated over a 1-year period at 3 institutions. We were able to classify all individuals using the OMT classification system.

Decker R.S., Koyama E., Pacifici M.

Limb synovial joints are intricate structures composed of articular cartilage, synovial membranes, ligaments and an articular capsule. Together, these tissues give each joint its unique shape, organization and biomechanical function. Articular cartilage itself is rather complex and organized in distinct zones, including the superficial zone that produces lubricants and contains stem/progenitor cells. For many years there has been great interest in deciphering the mechanisms by which the joints form and come to acquire such unique structural features and diversity. Decades ago, classic embryologists discovered that the first overt sign of joint formation at each prescribed limb site was the appearance of a dense and compact population of mesenchymal cells collectively called the interzone. Work carried out since then by several groups has provided evidence that the interzone cells actively participate in joint tissue formation over developmental time. This minireview provides a succinct but comprehensive description of the many important recent advances in this field of research. These include studies using various conditional reporter mice to genetically trace and track the origin, fate and possible function of joint progenitor cells; studies on the involvement and roles in signaling pathways and transcription factors in joint cell determination and functioning; and studies using advanced methods of gene expression analyses to uncover novel genetic determinants of joint formation and diversity. The overall advances are impressive, and the findings are not only of obvious interest and importance but also have major implications in the conception of future translational medicine tools to repair and regenerate defective, overused or aging joints.

Bornes T.D., Adesida A.B., Jomha N.M.

Articular cartilage has a limited capacity to repair following injury. Early intervention is required to prevent progression of focal traumatic chondral and osteochondral defects to advanced cartilage degeneration and osteoarthritis. Novel cell-based tissue engineering techniques have been proposed with the goal of resurfacing defects with bioengineered tissue that recapitulates the properties of hyaline cartilage and integrates into native tissue. Transplantation of mesenchymal stem cells (MSCs) is a promising strategy given the high proliferative capacity of MSCs and their potential to differentiate into cartilage-producing cells - chondrocytes. MSCs are historically harvested through bone marrow aspiration, which does not require invasive surgical intervention or cartilage extraction from other sites as required by other cell-based strategies. Biomaterial matrices are commonly used in conjunction with MSCs to aid cell delivery and support chondrogenic differentiation, functional extracellular matrix formation and three-dimensional tissue development. A number of specific transplantation protocols have successfully resurfaced articular cartilage in animals and humans to date. In the clinical literature, MSC-seeded scaffolds have filled a majority of defects with integrated hyaline-like cartilage repair tissue based on arthroscopic, histologic and imaging assessment. Positive functional outcomes have been reported at 12 to 48 months post-implantation, but future work is required to assess long-term outcomes with respect to other treatment modalities. Despite relatively positive outcomes, further investigation is required to establish a consensus on techniques for treatment of chondral and osteochondral defects with respect to cell source, isolation and expansion, implantation density, in vitro precultivation, and scaffold composition. This will allow for further optimization of MSC proliferation, chondrogenic differentiation, bioengineered cartilage integration, and clinical outcome.

Gao X., Yourick J.J., Sprando R.L.

The Tox21 program calls for transforming toxicology testing from traditional in vivo tests to less expensive and higher throughput in vitro methods. In developmental toxicology, a spectrum of alternative methods including cell line based tests has been developed. In particular, embryonic stem cells (ESCs) have received widespread attention as a promising alternative model for developmental toxicity assessment. Here, we characterized gene expression changes during mouse ESC differentiation and their modulation by developmental toxicants. C57BL/6 ESCs were allowed to differentiate spontaneously and RNA of vehicle controls was collected at 0, 24, 48, 72, 96, 120 and 168 h after embryoid body (EB) formation; RNA of compound-exposed EBs were collected at 24 h. Samples were hybridized to Affymetrix Mouse Gene 2.0 ST Array; using stringent cut-off criteria of Bonferroni-adjusted p2.0, a total of 1996 genes were found differentially expressed among the vehicle controls at different time points. Gene ontology (GO) analysis showed these regulated genes were mostly involved in differentiation-related processes such as development, morphogenesis, metabolism, cell differentiation, cell organization and biogenesis, embryonic development, and reproduction. Biomarkers of all three germ layers or of their derivative early cell types were identified in the gene list. Principal component analysis (PCA) based on these genes showed that the unexposed vehicle controls appeared in chronological order in the PCA plot, and formed a differentiation track when connected. Cultures exposed to thalidomide, monobutyl phthalate, or valproic acid deviated significantly from the differentiation track, manifesting the capacity of the differentiation track to identify the modulating effects of diverse developmental toxicants. The differentiation track defined in this study may be further exploited as a baseline for developmental toxicity testing, with compounds causing significant deviation from the differentiation track being predicted as potential developmental toxicants.

Cheng X., Wang G., Lee K., Yang X.

Glucocorticoids are important regulators of cell differentiation and mesenchymal cell lineage commitment during skeletogenesis. In clinical practice, it has been difficult to study the effects of glucocorticoids on target tissues because patients taking glucocorticoids often suffer from adverse skeletal effects. Dexamethasone (Dex) is a long-acting synthetic corticosteroid hormone that ranks amongst the most widely used prescribed drugs, and it is a powerful medication that is increasingly employed during the perinatal and neonatal periods. However, Dex is a potential teratogen. In particular, it has been claimed that Dex exposure during pregnancy can affect osteogenesis in the developing embryo, although this claim remains highly controversial. In this review, we summarize the published data from numerous clinical follow-up, animal-based and in vitro studies on the effects of Dex exposure on embryonic skeletogenesis. These studies indicate that Dex may adversely affect skeletal progenitor cells during development. In addition, Dex can exert a number of effects on bone growth at different developmental stages. We also discuss how glucocorticoids influence the BMP, FGF, Hedgehog and Wnt signaling pathways, which are key regulators of skeletogenesis in the embryo. A fuller understanding of the negative, and perhaps teratogenic, effects of Dex on skeletogenesis will have important implications for the routine use of Dex in clinical practice.

Herion N.J., Salbaum J.M., Kappen C.

Gastrulation is the process in which the three germ layers are formed that contribute to the formation of all major tissues in the developing embryo. We here review mouse genetic models in which defective gastrulation leads to mesoderm insufficiencies in the embryo. Depending on severity of the abnormalities, the outcomes range from incompatible with embryonic survival to structural birth defects, such as heart defects, spina bifida, or caudal dysgenesis. The combined evidence from the mutant models supports the notion that these congenital anomalies can originate from perturbations of mesoderm specification, epithelial-mesenchymal transition, and mesodermal cell migration. Knowledge about the molecular pathways involved may help to improve strategies for the prevention of major structural birth defects.

Brosens E., Ploeg M., van Bever Y., Koopmans A.E., IJsselstijn H., Rottier R.J., Wijnen R., Tibboel D., de Klein A.

Esophageal Atresia (EA) is a severe developmental defect of the foregut that presents with or without a Tracheo-Esophageal Fistula (TEF). The prevalence of EA/TEF over time and around the world has been relatively stable. EA/TEF is manifested in a broad spectrum of anomalies: in some patients it manifests as an isolated atresia or fistula, but in over half it affects several organ systems. While the associated malformations are often those of the VACTERL spectrum (Vertebral, Anorectal, Cardiac, Tracheo-Esophageal, Renal and Limb), many patients are affected by other malformations, such as microcephaly, micrognathia, pyloric stenosis, duodenal atresia, a single umbilical artery, and anomalies of the genitourinary, respiratory and gastrointestinal systems. Though EA/TEF is a genetically heterogeneous condition, recurrent genes and loci are sometimes affected. Tracheo-Esophageal (TE) defects are in fact a variable feature in several known single gene disorders and in patients with specific recurrent Copy Number Variations and structural chromosomal aberrations. At present, a causal genetic aberration can be identified in 11-12% of patients. In most, EA/TEF is a sporadic finding; the familial recurrence rate is low (1%). As this suggests that epigenetic and environmental factors also contribute to the disease, non-syndromic EA/TEF is generally believed to be a multifactorial condition. Several population-based studies and case reports describe a wide range of associated risks, including age, diabetes, drug use, herbicides, smoking and fetal alcohol exposure. The phenotypical and genetic heterogeneity seen in EA/TEF patients indicates not one underlying cause, but several. Unraveling the complex multifactorial and heterogeneous etiology of EA/TEF and associated features will require large cohorts of patients. Combined statistical analysis of component findings, genome sequencing, and genome wide association studies will elucidate new causal genetic defects and predisposing loci in the etiology within specific sub-populations. Improved knowledge of environmental risk factors, genetic predisposition and causal genetic syndromes may improve prediction and parental counseling, and prevent co-morbidity.

Aryanfar Y., Wu Y., Alcaraz J.L., Vidal R.P., Osman A.I., Manap A.S., Afzal S., Saikia T., Chen Z., Lu K., Rooney D.W., Yap P.

Pu S., Wang Z., Tang X., Wang D., Yang X., Jiang J., Deng Y., Xiang B., Yang J., Wang X., Guo X., Sun M., Wang B., Chen J.

Preaxial polydactyly (PPD) is a congenital limb malformation, previously reported to be caused primarily by variants in the ZRS and upstream preZRS regions. This study investigated genetic variations associated with PPD, focusing on point variants and copy number variations (CNVs) in the ZRS and preZRS regions. Comprehensive genetic analyses were conducted on 102 patients with PPD, including detailed clinical examinations and Sanger sequencing of the ZRS and preZRS regions. Additionally, real-time quantitative PCR (qPCR) was used to detect CNVs in the ZRS region. The evolutionary conservation and population frequencies of identified variants were also evaluated. Six point variants were identified, among which four are likely pathogenic novel variants: 93G > T (g.156584477G > T), 106G > A (g.156584464G > A), 278G > A (g.156584292G > A), and 409A > C (g.156585378A > C). Additionally, qPCR analysis revealed that 66.67% of patients exhibited ZRS duplications. Notably, these duplications were also present in cases with newly identified potential pathogenic point variants. These findings suggest the possible interaction of point variants in ZRS and preZRS through a common pathogenic mechanism, leading jointly to PPD. The findings expand the variant spectrum associated with non-syndromic polydactyly and highlight that, despite different classifications, anterior polydactyly caused by variants in ZRS and nearby regions may share common pathogenic mechanisms. The incorporation of various variant types in genetic screening can effectively enhance the rate of pathogenic variant detection and contribute to the cost-effectiveness of genetic testing for limb developmental defects, thereby promoting healthy births.

Fortin M.C., Szilagyi J.

With the advent of molecular and cellular toxicology, the field of drug safety evaluation has been undergoing an important paradigm shift, which has recently been further incentivized through legislation. In vitro approaches are regularly used for internal decision making and as a means to de-risk, or at least better understand, the safety profile of compounds via investigative toxicology studies. These approaches now have the potential to mitigate some of the blind spots of traditional in vivo approaches and bolster preclinical safety evaluation. For each major physiological system or function, namely, the heart, central nervous system, endocrine system, gastrointestinal system, hematopoietic system and immune function, kidney, liver, male and female reproductive systems and teratogenicity, in vitro approaches using cell lines, human induced pluripotent stem cell (hiPSC)-derived cells, and tissue preparations or organoids, and microphysiological systems (MPS) are discussed in order of increasing complexity to provide a comprehensive overview of the tools currently available. Because this is an emerging field, it is not possible to provide standard protocols and endpoints as these must be tailored to the specific in vitro model and target organ system, and therefore, potential uses, technical considerations, and possible drawbacks of recent models are discussed instead. When possible, assays that have been more broadly established and become part of standard practices are also presented. As in vitro toxicology approaches continue to be improved and refined, safety evaluation will become more robust and both companies and patients will benefit.

Toovey R., Coulston F., Shuttleworth H., Jelleff M., Villalon J., Anderson S., Shields N., Finnigan M., Lalor S., McGinley J.

Zhang B., Berilla J., Cho S., Somoza R.A., Welter J.F., Alexander P.E., Baskaran H.

AbstractInterzone/cavitation are key steps in early stage joint formation that have not been successfully developed in vitro. Further, current models of endochondral ossification, an important step in early bone formation, lack key morphology morphological structures such as microcavities found during development in vivo. This is possibly due to the lack of appropriate strategies for incorporating chemical and mechanical stimuli that are thought to be involved in joint development. We designed a bioreactor system and investigated the synergic effect of chemical stimuli (chondrogenesis‐inducing [CIM] and hypertrophy‐inducing medium [HIM]) and mechanical stimuli (flexion) on the growth of human mesenchymal stem cells (hMSCs) based linear aggregates under different conditions over 4 weeks of perfusion culture. Computational studies were used to evaluate tissue stress qualitatively. After harvesting, both Safranin‐O and hematoxylin & eosin (H&E) staining histology demonstrated microcavity structures and void structures in the region of higher stresses for tissue aggregates cultured only in HIM under flexion. In comparison to either HIM treatment or flexion only, increased glycosaminoglycan (GAG) content in the extracellular matrix (ECM) at this region indicates the morphological change resembles the early stage of joint cavitation; while decreased type II collagen (Col II), and increased type X collagen (Col X) and vascular endothelial growth factor (VEGF) with a clear boundary in the staining section indicates it resembles the early stage of ossification. Further, cell alignment analysis indicated that cells were mostly oriented toward the direction of flexion in high‐stress region only in HIM under flexion, resembling cell morphology in both joint cavitation and hypertrophic cartilage in growth plate. Collectively, our results suggest that flexion and HIM inhibit chondrogenesis and promote hypertrophy and development of microcavities that resemble the early stage of joint cavitation and endochondral ossification. We believe the tissue model described in this work can be used to develop in vitro models of joint tissue for applications such as pathophysiology and drug discovery.

Boudet-Berquier J., Demattei C., Guldner L., Gallay A., Manouvrier S., Botton J., Philippat C., Delva F., Bloch J., Semaille C., Odent S., Perthus I., Randrianaivo H., Babajko S., Barjat T., et. al.

Introduction: Between 2019–2021, facing public concern, a scientific expert committee (SEC) reanalysed suspected clusters of transverse upper limb reduction defects (TULRD) in three administrative areas in France, where initial investigations had not identified any risk exposure. We share here the national approach we developed for managing suspicious clusters of the same group of congenital anomalies occurring in several areas. Methods: The SEC analysed the medical records of TURLD suspected cases and performed spatiotemporal analyses on confirmed cases. If the cluster was statistically significant and included at least three cases, the SEC reviewed exposures obtained from questionnaires, environmental databases, and a survey among farmers living near to cases’ homes concerning their plant product use. Results: After case re-ascertainment, no statistically significant cluster was observed in the first administrative areas. In the second area, a cluster of four children born in two nearby towns over two years was confirmed, but as with the initial investigations, no exposure to a known risk factor explaining the number of cases in excess was identified. In the third area, a cluster including just two cases born the same year in the same town was confirmed. Discussion: Our experience highlights that in the event of suspicious clusters occurring in different areas of a country, a coordinated and standardised approach should be preferred.

Yuan Q., Liu Y.

Micro/nano plastics (M/NPs) are emerging pollutants that have extensively infiltrated various aspects of human life, posing a significant threat to the natural ecological systems. M/NPs can enter the digestive system through the oral cavity and accumulate in various organs. The current research on M/NPs primarily relies on model organisms, and there remains a dearth of direct evidence concerning the impact of M/NPs on human health. Commonly utilized specific two dimensional (2D) cultured cell lines exhibit substantial disparities in physiological functions when compared to multicellular tissues in vivo. The conduct of animal experiments is a time-consuming process, constrained by ethical considerations, and also confronted with interspecies variations. A significant breakthrough in biology is the development of organoids derived from stem cells. Intestinal organoids can mimic the complex structure and functionality of tissue, and can generate cell-cell and cell-matrix interactions that closely resemble physiological responses in the body. As a result, they provide a more accurate reflection of toxic effects and mechanisms, and hold great potential for applications in the environmental toxicology assessment. However, the current research on the toxic mechanisms of M/NPs using intestinal organoids is still in its early stages. The focus of this review is on the application of intestinal organoids in toxicology studies of M/NPs, assessing the correlation between M/NPs and diseases, as well as elucidating the molecular mechanisms underlying toxic effects. Ultimately, we present the challenges and potential solutions for utilizing intestinal organoids as models to evaluate M/NPs-induced toxicity, aiming to provide valuable insights for future research.

Cevik J., Salehi O., Gaston J., Rozen W.M.

Maternal smoking during pregnancy has been associated with adverse effects on foetal development, including congenital limb anomalies. This systematic review aimed to provide an updated assessment of the association between maternal smoking during pregnancy and the risk of congenital limb anomalies. A systematic search was conducted to identify relevant studies published up to February 2023. Studies reporting on the relationship between maternal smoking during pregnancy and congenital digital anomalies or congenital limb reduction defects were included. Two independent reviewers screened the studies, extracted data, and assessed the quality of the included studies. Meta-analyses were performed to estimate the pooled odds ratios with 95% confidence intervals using fixed and random-effects models. In total, 37 publications comprising 11 cohort and 26 case-control studies were included in the systematic review. The meta-analysis demonstrated a significant increased risk of congenital limb reduction defects (pooled OR: 1.27, 95% CI: 1.18–1.38) in infants born to mothers who smoked during pregnancy. Similarly, a significant relationship was observed for the development of polydactyly/syndactyly/adactyly when considered as a single group (pooled OR: 1.32, 95% CI: 1.25–1.40). Yet, in contrast, no significant association was observed when polydactyly (pooled OR: 1.06, 95% CI: 0.88–1.27) or syndactyly (pooled OR: 0.91, 95% CI: 0.77–1.08) were considered individually. This systematic review provides updated evidence of a significant relationship between maternal smoking during pregnancy and increased risk of congenital limb anomalies. These findings highlight the potential detrimental effects of smoking on foetal limb development and underscore the importance of smoking cessation interventions for pregnant women to mitigate these risks.

Rajesh V., Kokilavani A., Jayaseelan S., Gomathi S., Vishali K., Kumudhavalli M.V.

Worldwide, estimated counts of about 7.9 million children are born with serious birth defects. In addition to genetic factors, prenatal exposure to drugs and environmental toxicants represents a major contributing factor to congenital malformations. In earlier investigation, we explored cardiac malformation caused by valproic acid (VPA) during early developing stages of zebrafish. Since heart depends on mitochondrial fatty acid oxidative metabolism for energy demands in which carnitine shuttle has a major role, the present study aimed to investigate the effect of acetyl-L-carnitine (AC) against VPA-induced cardiac malformation in developing zebrafish. Initially, AC was subjected to toxicological evaluation, and two micromolar concentrations (25 µM and 50 µM) were selected for evaluation. A sub-lethal concentration of VPA (50 µM) was selected to induce cardiac malformation. The embryos were grouped and the drug exposures were made at 2.5 h post-fertilization (hpf). The cardiac development and functioning was monitored. A progressive decline in cardiac functioning was noted in group exposed to VPA 50 µM. At 96 hpf and 120 hpf, the morphology of heart was severely affected with the chambers which became elongated and string-like accompanied by histological changes. Acridine orange staining showed accumulation of apoptotic cells. Group exposed to VPA 50 µM with AC 50 µM showed a significant reduction in pericardial sac edema with morphological, functional and histological recovery in developing heart. Moreover, reduced number of apoptotic cells was noted. The improvement with AC might be due to restoration of carnitine homeostasis for cardiac energy metabolism in developing heart.

Fortin M.C., Szilagyi J.

With the advent of molecular and cellular toxicology, the field of drug safety evaluation has been undergoing an important paradigm shift, which has recently been further incentivized through legislation. In vitro approaches are regularly used for internal decision making and as a means to de-risk, or at least better understand, the safety profile of compounds via investigative toxicology studies. These approaches now have the potential to mitigate some of the blind spots of traditional in vivo approaches and bolster preclinical safety evaluation. For each major physiological system or function, namely, the heart, central nervous system, endocrine system, gastrointestinal system, hematopoietic system and immune function, kidney, liver, male and female reproductive systems and teratogenicity, in vitro approaches using cell lines, human induced pluripotent stem cell (hiPSC)-derived cells, and tissue preparations or organoids, and microphysiological systems (MPS) are discussed in order of increasing complexity to provide a comprehensive overview of the tools currently available. Because this is an emerging field, it is not possible to provide standard protocols and endpoints as these must be tailored to the specific in vitro model and target organ system, and therefore, potential uses, technical considerations, and possible drawbacks of recent models are discussed instead. When possible, assays that have been more broadly established and become part of standard practices are also presented. As in vitro toxicology approaches continue to be improved and refined, safety evaluation will become more robust and both companies and patients will benefit.

Burns N.G., Kardon G.

Structural birth defects are a common cause of abnormalities in newborns. While there are cases of structural birth defects arising due to monogenic defects or environmental exposures, many birth defects are likely caused by a complex interaction between genes and the environment. A structural birth defect with complex etiology is congenital diaphragmatic hernias (CDH), a common and often lethal disruption in diaphragm development. Mutations in more than 150 genes have been implicated in CDH pathogenesis. Although there is generally less evidence for a role for environmental factors in the etiology of CDH, deficiencies in maternal vitamin A and its derivative embryonic retinoic acid are strongly associated with CDH. However, the incomplete penetrance of CDH-implicated genes and environmental factors such as vitamin A deficiency suggest that interactions between genes and environment may be necessary to cause CDH. In this review, we examine the genetic and environmental factors implicated in diaphragm and CDH development. In addition, we evaluate the potential for gene-environment interactions in CDH etiology, focusing on the potential interactions between the CDH-implicated gene, Gata4, and maternal vitamin A deficiency.

Larson J.E.

Despite improved prenatal access to care and increased knowledge of fetal toxic agents, babies are still born with musculoskeletal deformities that require attention from a pediatric orthopedic surgeon. A teratogenic agent or congenital infection is most likely to cause birth defects during organogenesis between days 15 and 60 of gestation. However, avoiding exposure to teratogens and infections throughout pregnancy is critical to decrease the risk of orthopedic manifestations related to in utero exposures. This chapter will highlight the most common teratogens and congenital infections which can predispose a fetus to orthopedic manifestations such as neural tube defects, limb deformities, and cerebral palsy secondary to premature delivery.

Watson A.T., Carmona Baez A., Jima D., Reif D., Ding J., Roberts R., Kullman S.W.

Abstract Differentiation of multipotent mesenchymal stem cells into bone-forming osteoblasts requires strict coordination of transcriptional pathways. Aryl hydrocarbon receptor (AhR) ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been shown to alter osteoblast differentiation in vitro and bone formation in multiple developmental in vivo models. The goal of the present study was to establish a global transcriptomic landscape during early, intermediate, and apical stages of osteogenic differentiation in vitro in response to TCDD exposure. Human bone-derived mesenchymal stem cells (hBMSC) were cultured in growth media (GM), osteogenic differentiation media (ODM), or osteogenic differentiation media containing 10 nM TCDD (ODM+TCDD), thus enabling a comparison of the transcriptomic profiles of undifferentiated, differentiated, and differentiated -TCDD-exposed hBMSCs, respectively. In this test system, exposure to TCDD attenuated differentiation of hBMSCs into osteoblasts as evidenced by reduced alkaline phosphatase activity and mineralization. At various timepoints, we observed altered expression of genes that play a role in the Wnt, FGF, BMP/TGF-β developmental pathways, as well as pathways related to extracellular matrix organization and deposition. Reconstruction of gene regulatory networks with the iDREM analysis revealed modulation of transcription factors (TF) including POLR3G, NR4A1, RDBP, GTF2B, POU2F2 and ZEB1, which may putatively influence osteoblast differentiation and the requisite deposition and mineralization of bone extracellular matrix. We demonstrate that the combination of RNA-Seq data in conjunction with the iDREM regulatory model, captures the transcriptional dynamics underlying mesenchymal stem cell differentiation under different conditions in vitro. Model predictions are consistent with existing knowledge and provides a new tool to identify novel pathways and transcription factors that may facilitate a better understanding of the osteoblast differentiation process, perturbation by exogenous agents, and potential intervention strategies targeting those specific pathways.

Shah H., Cotofana S., Lifchez S.D., Bialowas C.

With the advent of the subspecialty of orthoplastic surgery, there has been a greater involvement of plastic surgeons in limb reconstruction, congenital anomalies, and salvage procedures. Understanding embryology helps plastic surgeons better understand how to reconstruct lower extremity congenital defects and how development of the lower limb affects what can be done to reconstruct the adult lower limb. There is significant discussion in the literature regarding embryology and development of craniofacial structures and the upper extremity; however, the development of the lower limb remains largely unexplored. This review aims to elucidate the embryologic development of the lower limb, congenital anomalies that may present to the plastic surgeon, and reconstructive options.

Schaeffer T., Canizares M.F., Wall L.B., Bohn D., Steinman S., Samora J., Manske M.C., Hutchinson D.T., Shah A.S., Bauer A.S., Bae D.S., Goldfarb C.A., Cook D.L.

Purpose Risk factors for congenital upper limb differences (CoULDs) are often studied at the general population level. The CoULD registry provides a unique opportunity to study prenatal risk factors within a large patient sample. Methods All patients enrolled between June 2014 and March 2020 in the prospective CoULD registry, a national multicenter database of patients diagnosed with a CoULD, were included in the analysis. We analyzed self-reported, prenatal risk factors, including maternal smoking, alcohol use, recreational drug use, prescription drug use, gestational diabetes mellitus (GDM), and gestational hypertension. The outcome measures included comorbid medical conditions, proximal involvement of limb difference, bilateral involvement, and additional orthopedic conditions. Multivariable logistic regression was used to analyze the effect of the risk factors, controlling for sex and the presence of a named syndrome. Results In total, 2,410 patients were analyzed, of whom 72% (1,734) did not have a self-reported risk factor. Among the 29% (676) who did have at least 1 risk factor, prenatal maternal prescription drug use was the most frequent (376/2,410; 16%). Maternal prescription drug use was associated with increased odds of patient medical comorbidities (odds ratio [OR] = 1.43, P = .02). Gestational diabetes mellitus was associated with increased odds of comorbid medical conditions (OR = 1.58, P = .04), additional orthopedic conditions (OR = 1.51, P = .04), and proximal involvement (OR = 1.52, P = .04). Overall, reporting 1 or more risk factors increased the odds of patient comorbid medical conditions (OR = 1.42, P < .001) and additional orthopedic conditions (OR = 1.25, P = .03). Conclusions Most caregivers (72%) did not report a risk factor during enrollment. However, reporting a risk factor was associated with patient medical and orthopedic comorbidities. Of note, GDM alone significantly increased the odds of both these outcome measures along with proximal limb differences. These findings highlight the ill-defined etiology of CoULDs but suggest that prenatal risk factors, especially GDM, are associated with a higher degree of morbidity. Type of study/level of evidence Prognostic III.