A mechanism for gene-environment interaction in the etiology of congenital scoliosis.

Congenital scoliosis, a lateral curvature of the spine caused by vertebral defects, occurs in approximately 1 in 1,000 live births. Here we demonstrate that haploinsufficiency of Notch signaling pathway genes in humans can cause this congenital abnormality. We also show that in a mouse model, the combination of this genetic risk factor with an environmental condition (short-term gestational hypoxia) significantly increases the penetrance and severity of vertebral defects. We demonstrate that hypoxia disrupts FGF signaling, leading to a temporary failure of embryonic somitogenesis. Our results potentially provide a mechanism for the genesis of a host of common sporadic congenital abnormalities through gene-environment interaction.

The origins and roles of osteoclasts in bone development, homeostasis and repair.

The mechanisms underlying bone development, repair and regeneration are reliant on the interplay and communication between osteoclasts and other surrounding cells. Osteoclasts are multinucleated monocyte lineage cells with resorptive abilities, forming the bone marrow cavity during development. This marrow cavity, essential to hematopoiesis and osteoclast-osteoblast interactions, provides a setting to investigate the origin of osteoclasts and their multi-faceted roles. This Review examines recent developments in the embryonic understanding of osteoclast origin, as well as interactions within the immune environment to regulate normal and pathological bone development, homeostasis and repair.

CRISPR-SID: Identifying EZH2 as a druggable target for desmoid tumors via in vivo dependency mapping.

Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/ß-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast, efficient, and penetrant genetic Xenopus tropicalis desmoid tumor models to identify and characterize drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 selection-mediated identification of dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double-strand break repair patterns to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1 as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent approach for in vivo mapping of tumor vulnerabilities and drug target identification.

Pro-Con Debate: Interdisciplinary Perspectives on Industry-Sponsored Research.

PRO: Nearly all new devices and drugs come from industry that provides two-thirds of the funding for medical research, and a much higher fraction of clinical research. Realistically, without corporate-funded studies, perioperative research would stagnate with little innovation and few new products. Opinions are ubiquitous and normal but do not constitute epidemiologic bias. Competent clinical research includes many protections against selection and measurement bias, and the publication process provides at least moderate protection against misinterpretation of results. Trial registries largely prevent selective data presentation. Sponsored trials are particularly protected against inappropriate corporate influence because they are usually codesigned with the US Food and Drug Administration, and analyses are based on formal predefined statistical plans, as well as being conducted with rigorous external monitoring. Novel products, which are essential for advances in clinical care, largely come from industry, and industry appropriately funds much of the required research. We should celebrate industry's contribution to improvements in clinical care. CON: While industry funding contributes to research and discovery, examples of industry-funded research demonstrate bias. In the setting of financial pressures and potential conflict of interest, bias can influence the type of study design, hypotheses being tested, rigor and transparency in data analysis, interpretation, as well as reporting of the results. Unlike public granting agencies, industry does not necessarily provide funding based on unbiased peer review following an open call for proposals. The focus on success can influence the choice of a comparator, which might not be ideal among the possible alternatives, the language used in the publication, and even the ability to publish. Unpublished negative trials can result in selected information being withheld from the scientific community and the public. Appropriate safeguards are needed to ensure that research addresses the most important and relevant questions, that results are available even when they do not support the use of a product produced by the funding company, that populations studied reflect the relevant patients, that the most rigorous approaches are applied, that studies have the appropriate power to address the question posed, and that conclusions are presented in an unbiased manner.

Intracellular biosynthesis of lipids and cholesterol by Scap and Insig in mesenchymal cells regulates long bone growth and chondrocyte homeostasis.

During enchondral ossification, mesenchymal cells express genes regulating the intracellular biosynthesis of cholesterol and lipids. Here, we have investigated conditional deletion of Scap or of Insig1 and Insig2 (Scap inhibits intracellular biosynthesis and Insig proteins activate intracellular biosynthesis). Mesenchymal condensation and chondrogenesis was disrupted in mice lacking Scap in mesenchymal progenitors, whereas mice lacking the Insig genes in mesenchymal progenitors had short limbs, but normal chondrogenesis. Mice lacking Scap in chondrocytes showed severe dwarfism, with ectopic hypertrophic cells, whereas deletion of Insig genes in chondrocytes caused a mild dwarfism and shortening of the hypertrophic zone. In vitro studies showed that intracellular cholesterol in chondrocytes can derive from exogenous and endogenous sources, but that exogenous sources cannot completely overcome the phenotypic effect of Scap deficiency. Genes encoding cholesterol biosynthetic proteins are regulated by Hedgehog (Hh) signaling, and Hh signaling is also regulated by intracellular cholesterol in chondrocytes, suggesting a feedback loop in chondrocyte differentiation. Precise regulation of intracellular biosynthesis is required for chondrocyte homeostasis and long bone growth, and these data support pharmacological modulation of cholesterol biosynthesis as a therapy for select cartilage pathologies.

Enchondromatosis and Growth Plate Development.

PURPOSE OF REVIEW: Enchondroma is a common cartilage benign tumor that develops from dysregulation of chondrocyte terminal differentiation during growth plate development. Here we provide an overview of recent progress in understanding causative mutations for enchondroma, dysregulated signaling and metabolic pathways in enchondroma, and the progression from enchondroma to malignant chondrosarcoma. RECENT FINDINGS: Several signaling pathways that regulate chondrocyte differentiation are dysregulated in enchondromas. Somatic mutations in the metabolic enzymes isocitrate dehydrogenase 1 and 2 (IDH1/2) are the most common findings in enchondromas. Mechanisms including metabolic regulation, epigenetic regulation, and altered signaling pathways play a role in enchondroma formation and progression. Multiple pathways regulate growth plate development in a coordinated manner. Deregulation of the process can result in chondrocytes failing to undergo differentiation and the development of enchondroma.

The Calpain Gene is Correlated With Metal-on-Metal Hip Replacement Failures.

BACKGROUND: Metal-on-metal (MOM) total hip arthroplasty is associated with unacceptable failure rates secondary to metal ion reactions. Efforts to identify which patients will go on to failure have been limited; recently, there has been a suggestion for a potential genetic basis for the increased risk of revision in MOM hip replacements (MOMHRs). The purpose of this study is to determine whether certain immunologic genotypes are predictive of the need for revision in patients with MOM total hip implants. METHODS: This is a case-control study of all patients undergoing primary MOMHR between September 2002 and January 2012 with a minimum of 5-year follow-up. Our investigational "case" cohort was comprised of patients who underwent revision for MOMHR for a reason other than infection. A single-nucleotide polymorphism (SNP) array analysis was performed to identify a potential genetic basis for failure. RESULTS: Thirty-two patients (15 case and 17 control) were included in our analysis. All patients in the revision group had a chief complain of pain; revision patients were more likely to have a posterior approach (P = .01) and larger head size (P = .04) than nonrevision patients. No patient or implant characteristics were independently associated with revision in a multivariate analysis. Patients with SNP kgp9316441 were identified as having an increased odds of revision for MOM failure (P < .001). CONCLUSION: This study identified an SNP, kgp9316441, encoding proteins associated with inflammation and macrophage activation. This SNP was associated with significantly increased odds of revision for MOMHR. Future studies are warranted to validate this gene target both in vitro and in vivo. LEVEL OF EVIDENCE: III.

Effects of chondroitin sulfate proteoglycan 4 (NG2/CSPG4) on soft-tissue sarcoma growth depend on tumor developmental stage.

Sarcomas, and the mesenchymal precursor cells from which they arise, express chondroitin sulfate proteoglycan 4 (NG2/CSPG4). However, NG2/CSPG4's function and its capacity to serve as a therapeutic target in this tumor type are unknown. Here, we used cells from human tumors and a genetically engineered autochthonous mouse model of soft-tissue sarcomas (STSs) to determine NG2/CSPG4's role in STS initiation and growth. Inhibiting NG2/CSPG4 expression in established murine and human STSs decreased tumor volume by almost two-thirds and cell proliferation rate by 50%. NG2/CSPG4 antibody immunotherapy in human sarcomas established as xenografts in mice similarly decreased tumor volume, and expression of a lentivirus blocking NG2/CSPG4 expression inhibited tumor cell proliferation and increased the latency of engraftment. Gene profiling showed that Ng2/Cspg4 deletion altered the expression of genes regulating cell proliferation and apoptosis. Surprisingly, Ng2/Cspg4 deletion at the time of tumor initiation resulted in larger tumors. Gene expression profiling indicated substantial down-regulation of insulin-like growth factor binding protein (Igfbp) genes when Ng2/Cspg4 is depleted at tumor initiation, but not when Ng2/Cspg4 is depleted after tumor initiation. Such differences may have clinical significance, as therapeutic targeting of a signaling pathway such as NG2/CSPG4 may have different effects on cell behavior with tumor progression. NG2/CSPG4 depletion has divergent effects, depending on the developmental stage of sarcoma. In established tumors, IGF signaling is active, and NG2 inhibition targets cell proliferation and apoptosis.

Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia.

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Δ14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.

The Role of the Immune Cells in Fracture Healing.

PURPOSE OF REVIEW: Bone fracture healing is a complex physiological process relying on numerous cell types and signals. Inflammatory factors secreted by immune cells help to control recruitment, proliferation, differentiation, and activation of hematopoietic and mesenchymal cells. Within this review we will discuss the functional role of immune cells as it pertains to bone fracture healing. In doing so, we will outline the cytokines secreted and their effects within the healing fracture callus. RECENT FINDINGS: Macrophages have been found to play an important role in fracture healing. These immune cells signal to other cells of the fracture callus, modulating bone healing. Cytokines and cellular signals within fracture healing continue to be studied. The findings from this work have helped to reinforce the importance of osteoimmunity in bone fracture healing. Owing to these efforts, immunomodulation is emerging as a potential therapeutic target to improve bone fracture healing.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Mechanism of hard-nanomaterial clearance by the liver.

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.

ß-Catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.

Tibial pseudarthrosis causes substantial morbidity in patients with neurofibromatosis type 1 (NF1). We studied tibial pseudarthrosis tissue from patients with NF1 and found elevated levels of ß-catenin compared to unaffected bone. To elucidate the role of ß-catenin in fracture healing, we used a surgically induced tibial fracture model in conditional knockout (KO) Nfl (Nf1(flox/flox)) mice. When treated with a Cre-expressing adenovirus (Ad-Cre), there was a localized knockdown of Nf1 in the healing fracture and a subsequent development of a fibrous pseudarthrosis. Consistent with human data, elevated ß-catenin levels were found in the murine fracture sites. The increased fibrous tissue at the fracture site was rescued by local treatment with a Wingless-type MMTV integration site (Wnt) antagonist, Dickkopf-1 (Dkk1). The murine pseudarthrosis phenotype was also rescued by conditional ß-catenin gene inactivation. The number of colony-forming unit osteoblasts (CFU-Os), a surrogate marker of undifferentiated mesenchymal cells able to differentiate to osteoblasts, correlated with the capacity to form bone at the fracture site. Our findings indicate that the protein level of ß-catenin must be precisely regulated for normal osteoblast differentiation. An up-regulation of ß-catenin in NF1 causes a shift away from osteoblastic differentiation resulting in a pseudarthrosis in vivo These results support the notion that pharmacological modulation of ß-catenin can be used to treat pseudarthrosis in patients with NF1.-Ghadakzadeh, S., Kannu, P., Whetstone, H., Howard A., Alman, B. A. ß-catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.

Specification of chondrocytes and cartilage tissues from embryonic stem cells.

Osteoarthritis primarily affects the articular cartilage of synovial joints. Cell and/or cartilage replacement is a promising therapy, provided there is access to appropriate tissue and sufficient numbers of articular chondrocytes. Embryonic stem cells (ESCs) represent a potentially unlimited source of chondrocytes and tissues as they can generate a broad spectrum of cell types under appropriate conditions in vitro. Here, we demonstrate that mouse ESC-derived chondrogenic mesoderm arises from a Flk-1(-)/Pdgfrα(+) (F(-)P(+)) population that emerges in a defined temporal pattern following the development of an early cardiogenic F(-)P(+) population. Specification of the late-arising F(-)P(+) population with BMP4 generated a highly enriched population of chondrocytes expressing genes associated with growth plate hypertrophic chondrocytes. By contrast, specification with Gdf5, together with inhibition of hedgehog and BMP signaling pathways, generated a population of non-hypertrophic chondrocytes that displayed properties of articular chondrocytes. The two chondrocyte populations retained their hypertrophic and non-hypertrophic properties when induced to generate spatially organized proteoglycan-rich cartilage-like tissue in vitro. Transplantation of either type of chondrocyte, or tissue generated from them, into immunodeficient recipients resulted in the development of cartilage tissue and bone within an 8-week period. Significant ossification was not observed when the tissue was transplanted into osteoblast-depleted mice or into diffusion chambers that prevent vascularization. Thus, through stage-specific manipulation of appropriate signaling pathways it is possible to efficiently and reproducibly derive hypertrophic and non-hypertrophic chondrocyte populations from mouse ESCs that are able to generate distinct cartilage-like tissue in vitro and maintain a cartilage tissue phenotype within an avascular and/or osteoblast-free niche in vivo.

Bone Marrow Stress Decreases Osteogenic Progenitors.

Age-related bone loss may be a result of declining levels of stem cells in the bone marrow. Using the Col2.3Δtk (DTK) transgenic mouse, osteoblast depletion was used as a source of marrow stress in order to investigate the effects of aging on osteogenic progenitors which reside in the marrow space. Five-month-old DTK mice were treated with one or two cycles of ganciclovir to conditionally ablate differentiated osteoblasts, whereas controls were saline-treated. Treatment cycles were two weeks in length followed by four weeks of recovery. All animals were sacrificed at 8 months of age; bone marrow stromal cells (BMSCs) were harvested for cell culture and whole bones were excised for bone quality assessment. Colony-forming unit (CFU) assays were conducted to investigate the osteogenic potential of BMSC in vitro, and RNA was extracted to assess the expression of osteoblastic genes. Bone quality assessments included bone histomorphometry, TRAP staining, microcomputed tomography, and biomechanical testing. Osteoblast depletion decreased CFU-F (fibroblast), CFU-ALP (alkaline phosphatase), and CFU-VK (von Kossa) counts and BMSC osteogenic capacity in cell culture. Ex vivo, there were no differences in bone mineral density of vertebrae or femurs between treatment groups. Histology showed a decrease in bone volume and bone connectivity with repeated osteoblast depletion; however, this was accompanied by an increase in bone formation rate. There were no notable differences in osteoclast parameters or observed bone marrow adiposity. We have developed a model that uses bone marrow stress to mimic age-related decrease in osteogenic progenitors. Our data suggest that the number of healthy BMSCs and their osteogenic potential decline with repeated osteoblast depletion. However, activity of the remaining osteoblasts increases to compensate for this loss in progenitor osteogenic potential.

Disruption of crosstalk between mesenchymal stromal and tumor cells in bone marrow as a therapeutic target to prevent metastatic bone disease.

Skeletal metastasis is a serious complication of many primary cancers. A common feature of tumor cells that metastasize to the bone marrow microenvironment is that they initiate a cascade of events, recruiting and presumably/potentially altering the phenotype of bone marrow mesenchymal stromal cells (MSC) to produce an environment that allows for tumor growth and in some cases, drug-resistant dormancy of latent cancer cells. Consequently the MSC population can contribute to metastatic disease through several distinct mechanisms by differentiating into cancer-associated fibroblasts (CAFs). Understanding the expression and epigenetic changes that occur as normal MSCs become associated with metastatic tumors would reveal possible therapeutic targets for treating skeletal metastasis.

Involvement and targeted intervention of dysregulated Hedgehog signaling in osteosarcoma.

BACKGROUND: During development, the Hedgehog pathway plays important roles regulating the proliferation and differentiation of chondrocytes, providing a template for growing bone. In this study, the authors investigated the components of dysregulated Hedgehog signaling as potential therapeutic targets for osteosarcoma. METHODS: Small-molecule agonists and antagonists that modulate the Hedgehog pathway at different levels were used to investigate the mechanisms of dysregulation and the efficacy of Hedgehog blockade in osteosarcoma cell lines. The inhibitory effect of a small-molecule Smoothened (SMO) antagonist, IPI-926 (saridegib), also was examined in patient-derived xenograft models. RESULTS: An inverse correlation was identified in osteosarcoma cell lines between endogenous glioma-associated oncogene 2 (GLI2) levels and Hedgehog pathway induction levels. Cells with high levels of GLI2 were sensitive to GLI inhibition, but not SMO inhibition, suggesting that GLI2 overexpression may be a mechanism of ligand-independent activation. In contrast, cells that expressed high levels of the Hedgehog ligand gene Indian hedgehog (IHH) and the target genes patched 1 (PTCH1) and GLI1 were sensitive to modulation of both SMO and GLI, suggesting ligand-dependent activation. In 2 xenograft models, active autocrine and paracrine, ligand-dependent Hedgehog signaling was identified. IPI-926 inhibited the Hedgehog signaling interactions between the tumor and the stroma and demonstrated antitumor efficacy in 1 of 2 ligand-dependent models. CONCLUSIONS: The current results indicate that both ligand-dependent and ligand-independent Hedgehog dysregulation may be involved in osteosarcoma. It is the first report to demonstrate Hedgehog signaling crosstalk between the tumor and the stroma in osteosarcoma. The inhibitory effect of IPI-926 warrants additional research and raises the possibility of using Hedgehog pathway inhibitors as targeted therapeutics to improve treatment for osteosarcoma.

Kif7 promotes hedgehog signaling in growth plate chondrocytes by restricting the inhibitory function of Sufu.

Proper regulation of Indian hedgehog (Ihh) signaling is vital for chondrocyte proliferation and differentiation in the growth plate. Its dysregulation causes skeletal dysplasia, osteoarthritis or cartilaginous neoplasia. Here, we show that Suppressor of fused (Sufu) and Kif7 are essential regulators of Ihh signaling. While Sufu acts as a negative regulator of Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in the turnover of Sufu and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dose of Sufu restores normal hedgehog pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory activity of Sufu. Furthermore, Kif7 also inhibits Gli transcriptional activity in the chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and -independent mechanisms.

Are quantum dots toxic? Exploring the discrepancy between cell culture and animal studies.

Despite significant interest in developing quantum dots (QDs) for biomedical applications, many researchers are convinced that QDs will never be used for treating patients because of their potential toxicity. The perception that QDs are toxic is rooted in two assumptions. Cadmium-containing QDs can kill cells in culture. Many researchers then assume that because QDs are toxic to cells, they must be toxic to humans. In addition, many researchers classify QDs as a homogeneous group of materials. Therefore, if CdSe QDs are harmful, they extrapolate this result to all QDs. Though unsubstantiated, these assumptions continue to drive QD research. When dosing is physiologically appropriate, QD toxicity has not been demonstrated in animal models. In addition, QDs are not uniform: each design is a unique combination of physicochemical properties that influence biological activity and toxicity. In this Account, we summarize key findings from in vitro and in vivo studies, explore the causes of the discrepancy in QD toxicological data, and provide our view of the future direction of the field. In vitro and in vivo QD studies have advanced our knowledge of cellular transport kinetics, mechanisms of QD toxicity, and biodistribution following animal injection. Cell culture experiments have shown that QDs undergo design-dependent intracellular localization and they can cause cytotoxicity by releasing free cadmium into solution and by generating free radical species. In animal experiments, QDs preferentially enter the liver and spleen following intravascular injection, undergo minimal excretion if larger than 6 nm, and appear to be safe to the animal. In vitro and in vivo studies show an apparent discrepancy with regard to toxicity. Dosing provides one explanation for these findings. Under culture conditions, a cell experiences a constant QD dose, but the in vivo QD concentration can vary, and the organ-specific dose may not be high enough to induce detectable toxicity. Because QDs are retained within animals, long-term toxicity may be a problem but has not been established. Future QD toxicity studies should be standardized and systematized because methodological variability in the current body of literature makes it difficult to compare and contrast results. We advocate the following steps for consistent, comparable toxicology data: (a) standardize dose metrics, (b) characterize QD uptake concentration, (c) identify in vitro models that reflect the cells QDs interact with in vivo, and (d) use multiple assays to determine sublethal toxicity and biocompatibility. Finally, we should ask more specific toxicological questions. For example: "At what dose are 5 nm CdSe QDs that are stabilized with mercaptoacetic acid and conjugated to the antibody herceptin toxic to HeLa cells?" rather than "Are QDs toxic?" QDs are still a long way from realizing their potential as a medical technology. Modifying the current QD toxicological research paradigm, investigating toxicity in a case-by-case manner, and improving study quality are important steps in identifying a QD formulation that is safe for human use.

Cutaneous wound healing: recruiting developmental pathways for regeneration.

Following a skin injury, the damaged tissue is repaired through the coordinated biological actions that constitute the cutaneous healing response. In mammals, repaired skin is not identical to intact uninjured skin, however, and this disparity may be caused by differences in the mechanisms that regulate postnatal cutaneous wound repair compared to embryonic skin development. Improving our understanding of the molecular pathways that are involved in these processes is essential to generate new therapies for wound healing complications. Here we focus on the roles of several key developmental signaling pathways (Wnt/ß-catenin, TGF-ß, Hedgehog, Notch) in mammalian cutaneous wound repair, and compare this to their function in skin development. We discuss the varying responses to cutaneous injury across the taxa, ranging from complete regeneration to scar tissue formation. Finally, we outline how research into the role of developmental pathways during skin repair has contributed to current wound therapies, and holds potential for the development of more effective treatments.