Antoine L. Carre

Estrogen/Estrogen Receptor Alpha Signaling in Mouse Posterofrontal Cranial Suture Fusion

Background While premature suture fusion, or craniosynostosis, is a relatively common condition, the cause is often unknown. Estrogens are associated with growth plate fusion of endochondral bones. In the following study, we explore the previously unknown significance of estrogen/estrogen receptor signaling in cranial suture biology. Methodology/Principal Findings Firstly, estrogen receptor (ER) expression was examined in physiologically fusing (posterofrontal) and patent (sagittal) mouse cranial sutures by quantitative RT-PCR. Next, the cranial suture phenotype of ER alpha and ER beta knockout (αERKO, βERKO) mice was studied. Subsequently, mouse suture-derived mesenchymal cells (SMCs) were isolated; the effects of 17-β estradiol or the estrogen antagonist Fulvestrant on gene expression, osteogenic and chondrogenic differentiation were examined in vitro. Finally, in vivo experiments were performed in which Fulvestrant was administered subcutaneously to the mouse calvaria. Results showed that increased ERα but not ERβ transcript abundance temporally coincided with posterofrontal suture fusion. The αERKO but not βERKO mouse exhibited delayed posterofrontal suture fusion. In vitro, addition of 17-β estradiol enhanced both osteogenic and chondrogenic differentiation in suture-derived mesenchymal cells, effects reversible by Fulvestrant. Finally, in vivo application of Fulvestrant significantly diminished calvarial osteogenesis, inhibiting suture fusion. Conclusions/Significance Estrogen signaling through ERα but not ERβ is associated with and necessary for normal mouse posterofrontal suture fusion. In vitro studies suggest that estrogens may play a role in osteoblast and/or chondrocyte differentiation within the cranial suture complex.

Calcium-Based Nanoparticles Accelerate Skin Wound Healing

Introduction Nanoparticles (NPs) are small entities that consist of a hydroxyapatite core, which can bind ions, proteins, and other organic molecules from the surrounding environment. These small conglomerations can influence environmental calcium levels and have the potential to modulate calcium homeostasis in vivo. Nanoparticles have been associated with various calcium-mediated disease processes, such as atherosclerosis and kidney stone formation. We hypothesized that nanoparticles could have an effect on other calcium-regulated processes, such as wound healing. In the present study, we synthesized pH-sensitive calcium-based nanoparticles and investigated their ability to enhance cutaneous wound repair. Methods Different populations of nanoparticles were synthesized on collagen-coated plates under various growth conditions. Bilateral dorsal cutaneous wounds were made on 8-week-old female Balb/c mice. Nanoparticles were then either administered intravenously or applied topically to the wound bed. The rate of wound closure was quantified. Intravenously injected nanoparticles were tracked using a FLAG detection system. The effect of nanoparticles on fibroblast contraction and proliferation was assessed. Results A population of pH-sensitive calcium-based nanoparticles was identified. When intravenously administered, these nanoparticles acutely increased the rate of wound healing. Intravenously administered nanoparticles were localized to the wound site, as evidenced by FLAG staining. Nanoparticles increased fibroblast calcium uptake in vitro and caused contracture of a fibroblast populated collagen lattice in a dose-dependent manner. Nanoparticles also increased the rate of fibroblast proliferation. Conclusion Intravenously administered, calcium-based nanoparticles can acutely decrease open wound size via contracture. We hypothesize that their contraction effect is mediated by the release of ionized calcium into the wound bed, which occurs when the pH-sensitive nanoparticles disintegrate in the acidic wound microenvironment. This is the first study to demonstrate that calcium-based nanoparticles can have a therapeutic benefit, which has important implications for the treatment of wounds.

Retinoic Acid Enhances Osteogenesis in Cranial Suture–Derived Mesenchymal Cells: Potential Mechanisms of Retinoid-Induced Craniosynostosis

BACKGROUND In utero retinoid exposure results in numerous craniofacial malformations, including craniosynostosis. Although many malformations associated with retinoic acid syndrome are associated with neural crest defects, the specific mechanisms of retinoid-induced craniosynostosis remain unclear. The authors used the culture of mouse cranial suture-derived mesenchymal cells to probe the potential cellular mechanisms of this teratogen to better elucidate mechanisms of retinoid-induced suture fusion. METHODS Genes associated with retinoid signaling were assayed in fusing (posterofrontal) and patent (sagittal, coronal) sutures by quantitative real-time polymerase chain reaction. Cultures of mouse suture-derived mesenchymal cells from the posterofrontal suture were established from 4-day-old mice. Cells were cultured with all-trans retinoic acid (1 and 5 muM). Proliferation, osteogenic differentiation, and specific gene expression were assessed. RESULTS Mouse sutures were found to express genes necessary for retinoic acid synthesis, binding, and signal transduction, demonstrated by quantitative real-time polymerase chain reaction (Raldh1, Raldh2, Raldh3, and Rbp4). These genes were not found to be differentially expressed in fusing as compared with patent cranial sutures in vivo. Addition of retinoic acid enhanced the osteogenic differentiation of suture-derived mesenchymal cells in vitro, including up-regulation of alkaline phosphatase activity and Runx2 expression. Contemporaneously, cellular proliferation was repressed, as shown by proliferative cell nuclear antigen expression. The pro-osteogenic effect of retinoic acid was accompanied by increased gene expression of several hedgehog and bone morphogenetic protein ligands. CONCLUSIONS Retinoic acid represses proliferation and enhances osteogenic differentiation of suture-derived mesenchymal cells. These in vitro data suggest that retinoid exposure may lead to premature cranial suture fusion by means of enhanced osteogenesis and hedgehog and bone morphogenetic protein signaling.

Solvent microenvironments and copper binding alters the conformation and toxicity of a prion fragment.

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu2+ ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu2+ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu2+ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu2+ ions, owing to binding affinity of copper towards histidine moiety present in the peptide.

Fetal mouse skin heals scarlessly in a chick chorioallantoic membrane model system.

AbstractIn mammals, the early-gestation fetus has the regenerative ability to heal skin wounds without scar formation. This observation was first reported more than 3 decades ago, and has been confirmed in a number of in vivo animal models. Although an intensive research effort has focused on unraveling the mechanisms underlying scarless fetal wound repair, no suitable model of in vitro fetal skin healing has been developed. In this article, we report a novel model for the study of fetal wound healing. Fetal skin from gestational day 16.5 Balb/c mice (total gestation, 20 days) was grafted onto the chorioallantoic membrane of 12-day-old chicken embryos and cultured for up to 7 days. At 48 hours postengraftment, circular wounds (diameter = 1 mm) were made in the fetal skin using a rotating titanium sapphire laser (N = 45). The tissue was examined daily by visual inspection to look for signs of infection and ischemia. The grafts and the surrounding host tissue were examined histologically. In all fetal skin grafts, the wounds completely reepithelialized by postinjury day 7, with regeneration of the dermis. Fetal mouse skin xenografts transplanted onto the chorioallantoic membrane of fertilized chicken eggs provides a useful model for the study of fetal wound healing. This model can be used as an adjunct to traditional in vivo mammalian models of fetal repair.

β-catenin-dependent wnt signaling: A pathway in acute cutaneous wounding

Background: Acute wound healing is a dynamic process that results in the formation of scar tissue. The mechanisms of this process are not well understood; numerous signaling pathways are thought to play a major role. Here, the authors have identified &bgr;-catenin–dependent Wnt signaling as an early acute-phase reactant in acute wound healing and scar formation. Methods: The authors created 6-mm full-thickness excisional cutaneous wounds on adult &bgr;-catenin–dependent Wnt signal (BAT-gal) reporter mice. The expression of canonical Wnt after wounding was analyzed using X-gal staining and quantitative real-time polymerase chain reaction. Next, recombinant mouse Wnt3a (rmWnt3a) was injected subcutaneously to the wound edge, daily. The mice were killed at stratified time points, up to 15 days after injury. Histologic analysis, quantitative real-time polymerase chain reaction, and Western blot were performed. Results: Numerous individual Wnt ligands increased in expression after wounding, including Wnt3a, Wnt4, Wnt10a, and Wnt11. A specific pattern of Wnt activity was observed, localized to the hair follicle and epidermis. Mice injected with rmWnt3a exhibited faster wound closure, increased scar size, and greater expression of fibroblast growth factor receptor-2 and type I collagen. Conclusions: The authors’ data suggest that &bgr;-catenin–dependent Wnt signaling expression increases shortly after cutaneous wounding, and exogenous rmWnt3a accelerates reepithelialization, wound matrix maturation, and scar formation. Future experiments will focus on the intersection of Wnt signaling and other known profibrotic cytokines.

Invited commentary: Discharge prevalence estimation, high altitude and microtia in Ecuadorian patients

Microtia is a developmental malformation of the external ear, characterized by a small, abnormally shaped auricle [1]. The estimated prevalence varies between 0.8 and 4.2 per 10,000 births depending on the population [2]. Microtia is most often unilateral, observed more frequently in men, and is associated with atresia or stenosis of the ear canal in 55–93% of patients [3]. The most common system for grading of severity for this variable congenital anomaly is the Marx classification; there is a strong correlation between the degree of microtia and the presence of middle ear anomalies [4]. The better understanding of the etiopathogenic factors associated with microtia may lead toward the accrual of new techniques for the screening, prevention, and improved treatment of this common congenital anomaly. Gonzalez-Andrade et al, in their manuscript “Discharge prevalence estimation, high altitude and microtia in Ecuadorian patients” have attempted to more ful-

Complex reconstruction of the lower extremity following sarcoma resection: a literature review

While amputation was traditionally the only option available for patients with sarcomas of the extremities, chemotherapy, radiation, and advances in microsurgical technique have allowed many patients to undergo limb-salvaging procedures. Given the low incidence and heterogeneity of these tumors, there is currently no standard treatment algorithm for limb reconstruction after large sarcoma resection. Thus, we systematically reviewed the various types of free tissue transfer used for the reconstruction of lower limbs after sarcoma resection. Techniques were described based on anatomic location. This literature review supports free tissue transfer as a safe and acceptable modality for reconstruction after sarcoma resection of the lower limb. It allows for the application of healthy vascularized tissue to the defect while also providing freedom of flap positioning. Flap choice is dependent on tumor and defect size, tissue type and function, as well as donor site availability.

3: COORDINATE OSTEOGENIC DIFFERENTIATION VIA RETINOIC ACID, SONIC HEDGEHOG AND BONE MORPHOGENETIC PROTEIN SIGNALING PATHWAYS IN SUTURE-DERIVED MESENCHYMAL CELLS: INSIGHTS INTO SPORADIC CRANIOSYNOSTOSIS

Introduction: Despite much research, the mechanisms of sporadic craniosynostosis, or premature suture fusion, remain poorly understood. The mouse model of physiologic suture fusion provides an excellent framework for the study of cranial suture biology. Our laboratory has extensively utilized the isolation and culture of suture-derived mesenchymal cells to probe potential signaling cascades of importance in suture ossification. Here, we examined the potential coordinate roles of retinoic acid (RA), Hedgehog, and Bone Morphogenetic Protein (BMP) signaling in suture ossification and fusion.