Flatfoot in children: anatomy of decision making

Concern about a child’s foot posture is a common reason for frequent consultations for an array of health care professionals; sports medicine specialists are often the first to recognize and advise on foot pathology. In the decision making process, it is essential to distinguish between the different types of flatfoot deformity: paediatric or adult, congenital or acquired, flexible or rigid. Although flatfoot in children is a common finding, evidence for the techniques of the reliable and reproducible assessment of the foot posture is scant. This general review presents the factors involved in the forming and supporting of the foot arches, discusses the protocols useful in the evaluation of the foot posture, and indicates how to differentiate between flatfoot cases needing treatment and cases that need only reassurance

A low-cost scalable 3D-printed sample-holder for agitation-based decellularization of biological tissues

Decellularized extracellular matrix is one of the most promising biological scaffold supporting in vitro tissue growth and in vivo tissue regeneration in both preclinical research and clinical practice. In case of thick tissues or even organs, conventional static decellularization methods based on chemical or enzymatic treatments are not effective in removing the native cellular material without affecting the extracellular matrix. To overcome this limitation, dynamic decellularization methods, mostly based on perfusion and agitation, have been proposed. In this study, we developed a low-cost scalable 3D-printed sample-holder for agitation-based decellularization purposes, designed for treating multiple specimens simultaneously and for improving efficiency, homogeneity and reproducibility of the decellularization treatment with respect to conventional agitation-based approaches. In detail, the proposed sample-holder is able to house up to four specimens and, immersed in the decellularizing solution within a beaker placed on a magnetic stirrer, to expose them to convective flow, enhancing the solution transport through the specimens while protecting them. Computational fluid dynamics analyses were performed to investigate the fluid phenomena establishing within the beaker and to support the sample-holder design. Exploratory biological tests performed on human skin specimens demonstrated that the sample-holder reduces process duration and increases treatment homogeneity and reproducibility

Flatfoot in children: anatomy of decision making

Concern about a child’s foot posture is a common reason for frequent consultations for an array of health care professionals; sports medicine specialists are often the first to recognize and advise on foot pathology. In the decision making process, it is essential to distinguish between the different types of flatfoot deformity: paediatric or adult, congenital or acquired, flexible or rigid. Although flatfoot in children is a common finding, evidence for the techniques of the reliable and reproducible assessment of the foot posture is scant. This general review presents the factors involved in the forming and supporting of the foot arches, discusses the protocols useful in the evaluation of the foot posture, and indicates how to differentiate between flatfoot cases needing treatment and cases that need only reassurance

Localization and origin of cardiac CD117-positive cells: identification of a population of epicardially-derived cells in adult human heart

During heart morphogenesis, epicardial cells undergo epithelial-mesenchymal transition giving origin to a population of epicardially derived cells that play a crucial role in the development of most cardiac cell lineages. Considering the hypothesis that epithelial-mesenchymal transition of epicardial mesothelium can generate cardiac primitive cells in the adult heart, we have examined in vivo and in vitro the epicardium and subepicardium of normal human adult hearts and of pathological hearts from patients with chronic ischemic heart failure for the presence of CD117positive cells with epithelial and mesenchymal markers expression. The number of CD117positive cells increased significantly in the subepicardium of pathological hearts and sloped down towards myocardium, remaining still elevated with respect to normal hearts. While cells with typical epithelial proteins expression formed an intact layer on the surface of the normal hearts, CD117positive cells were localized mainly in the subepicardium and expressed mesenchymal markers in the pathological hearts. Epithelial-mesenchymal transition, induced in vitro by several growth factors known to accumulate in the ischemic myocardium, gave origin to epicardiallyderived cells with CD117 expression. These data support the hypothesis of epicardial origin of cardiac primitive cells in the adult human heart

Localization and origin of cardiac CD117-positive cells: identification of a population of epicardially-derived cells in adult human heart.

During heart morphogenesis, epicardial cells undergo epithelial-mesenchymal transition giving origin to a population of epicardially derived cells that play a crucial role in the development of most cardiac cell lineages. Considering the hypothesis that epithelial-mesenchymal transition of epicardial mesothelium can generate cardiac primitive cells in the adult heart, we have examined in vivo and in vitro the epicardium and subepicardium of normal human adult hearts and of pathological hearts from patients with chronic ischemic heart failure for the presence of CD117positive cells with epithelial and mesenchymal markers expression. The number of CD117positive cells increased significantly in the subepicardium of pathological hearts and sloped down towards myocardium, remaining still elevated with respect to normal hearts. While cells with typical epithelial proteins expression formed an intact layer on the surface of the normal hearts, CD117positive cells were localized mainly in the subepicardium and expressed mesenchymal markers in the pathological hearts. Epithelial-mesenchymal transition, induced in vitro by several growth factors known to accumulate in the ischemic myocardium, gave origin to epicardiallyderived cells with CD117 expression. These data support the hypothesis of epicardial origin of cardiac primitive cells in the adult human heart

Doxorubicin causes depletion of cardiac primitive cell pool that may add to the mechanisms of doxorubicin-mediated delayed cardiotoxicity.

Doxorubicin is one of the most active anti-cancer drugs, widely used for the treatment of several malignancies, however its clinical implementation is limited by high incidence of cardiovascular events, most serious of them being the cardiomyopathy followed by congestive heart failure. Known studies of the mechanisms of doxorubicin toxicity focused on cardiomyocyte damage. But then cardiac primitive cells are considered to provide the adult heart with a substantial growth reserve determining the function of the heart throughout life. The role of this cell population in the myocardial response to different pathologic stimuli has been documented in animals and humans, suggesting cardiac primitive cells as possible pathophysiologic target in cardiac diseases. We advance the hypothesis that cardiotoxicity of doxorubicin may be due to its effects on cardiac primitive cells. Hence, we examined doxorubicin toxicity on this cell population. CD117-positive cells isolated from adult human normal atria were incubated with increasing concentrations of doxorubicin hydrochloride (0.1, 0.5 and 1μM), followed by the evaluation of proliferation and apoptosis after 12, 24, 48 and 60 hours. Proliferation was evaluated by BrdU incorporation and its rate dropped from 12.84±1.83% (control, n=4) to 2.41±0.27% (24 hours, 1μM, n=4, p<0,05). Immunoblotting of proteins from cardiac primitive cells lysate indicated the upregulation of cyclin B1 paired with reduced expression of phospho-cdk1 and increased expression of p27, corresponding to the presence of cell cycle block at G2/M transition. The expression of phospho-p53 (Ser15) the ratio phosphop53/total p53 increased 2.1-fold and 1.2-fold (p<0,05), respectively. Apoptosis was evidentiated by the detection of early mitochondrial potential disruption, followed by 2,4-fold increase in caspase-3 activity (4.8x10e-3 μmol pnitroaniline/min/ml in control vs 11.5x10e-3 at 48 hours). With respect to control, apoptotic cells were 6.2-fold and 10.4-fold (p<0,05) more numerous after 12 hours and 48 hours of incubation with 1μM of doxorubicin, respectively. Taking into consideration the known mechanism of doxorubicin cardiotoxicity, namely oxidative stress, we observed reduced expression of catalase and manganese superoxide dismutase in cardiac primitive cells after 36 hours of incubation with 1μM doxorubicin. In conclusion, doxorubicin has profound effects on cardiac primitive cell proliferation and survival. The depletion of CD117-positive cardiac primitive cell pool may add to the mechanisms of doxorubicin-mediated delayed cardiotoxicity

Optimization of Human Heart Decellularization Method

Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to 40x28x6mm, to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two-step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation

Optimization of Human Heart Decellularization Method

Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to 40x28x6mm, to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two-step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation

Fibroblast Looks Like a Mesenchymal Stem Cell and Talks Like a Mesenchymal Stem Cell: is It a Mesenchymal Stem Cell in Disguise?

Induced Pluripotent Stem Cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Fibroblast (FB) is the adult somatic cell most commonly used for reprogramming and is defined by spindle-shape morphology, plastic-adherence and expression of several markers. Nonetheless, such properties also define Mesenchymal Stem Cells (MSCs), that also share with dermal FB the ability to differentiate into osteoblasts, adipocytes and chondroblasts. Moving from the hypothesis that the most striking difference between FBs and MSCs is in the name, we isolated and cultured human dermal and visceral FBs from tissue waste fragments of patients undergoing surgical procedures, and evaluated their morphology and expression of markers typical of MSCs, at gene and protein level. Additionally, we analyzed synthesis and release of specific growth factors in culture medium and, finally, we tested FB ability to differentiate into osteoblasts, adipocytes and chondroblasts. Interestingly, all FBs in culture adhered to plastic culture dishes, expressed markers typical of MSCs, like CD90, CD105, CD146,SSEA-4, ECM2, ID-1, and were morphologically undistinguishable. As reported for MSCs, FBs released EGF, FGF-4, GDNF, HGF, IGF, TGF-b and VEGF, and were capable of differentiating into cells of mesodermal origin. Our findings raise the question of whether FBs and MSCs are not admittedly the same population.We might infer that previously described differences are due to different stages of differentiation, which also account for reports on high variability in both expression of mesenchymal markers by MSCs and efficiency of FB reprogramming to iPSCs

Fibroblast Looks Like a Mesenchymal Stem Cell and Talks Like a Mesenchymal Stem Cell: is It a Mesenchymal Stem Cell in Disguise?

Induced Pluripotent Stem Cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Fibroblast (FB) is the adult somatic cell most commonly used for reprogramming and is defined by spindle-shape morphology, plastic-adherence and expression of several markers. Nonetheless, such properties also define Mesenchymal Stem Cells (MSCs), that also share with dermal FB the ability to differentiate into osteoblasts, adipocytes and chondroblasts. Moving from the hypothesis that the most striking difference between FBs and MSCs is in the name, we isolated and cultured human dermal and visceral FBs from tissue waste fragments of patients undergoing surgical procedures, and evaluated their morphology and expression of markers typical of MSCs, at gene and protein level. Additionally, we analyzed synthesis and release of specific growth factors in culture medium and, finally, we tested FB ability to differentiate into osteoblasts, adipocytes and chondroblasts. Interestingly, all FBs in culture adhered to plastic culture dishes, expressed markers typical of MSCs, like CD90, CD105, CD146,SSEA-4, ECM2, ID-1, and were morphologically undistinguishable. As reported for MSCs, FBs released EGF, FGF-4, GDNF, HGF, IGF, TGF-b and VEGF, and were capable of differentiating into cells of mesodermal origin. Our findings raise the question of whether FBs and MSCs are not admittedly the same population.We might infer that previously described differences are due to different stages of differentiation, which also account for reports on high variability in both expression of mesenchymal markers by MSCs and efficiency of FB reprogramming to iPSCs