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

Decellularized Human Dermal Matrix as a Biological Scaffold for Cardiac Repair and Regeneration.

The complex and highly organized environment in which cells reside consists primarily of the extracellular matrix (ECM) that delivers biological signals and physical stimuli to resident cells. In the native myocardium, the ECM contributes to both heart compliance and cardiomyocyte maturation and function. Thus, myocardium regeneration cannot be accomplished if cardiac ECM is not restored. We hypothesize that decellularized human skin might make an easily accessible and viable alternate biological scaffold for cardiac tissue engineering (CTE). To test our hypothesis, we decellularized specimens of both human skin and human myocardium and analyzed and compared their composition by histological methods and quantitative assays. Decellularized dermal matrix was then cut into 600-mm-thick sections and either tested by uniaxial tensile stretching to characterize its mechanical behavior or used as three-dimensional scaffold to assess its capability to support regeneration by resident cardiac progenitor cells (hCPCs) in vitro. Histological and quantitative analyses of the dermal matrix provided evidence of both effective decellularization with preserved tissue architecture and retention of ECM proteins and growth factors typical of cardiac matrix. Further, the elastic modulus of the dermal matrix resulted comparable with that reported in literature for the human myocardium and, when tested in vitro, dermal matrix resulted a comfortable and protective substrate promoting and supporting hCPC engraftment, survival and cardiomyogenic potential. Our study provides compelling evidence that dermal matrix holds promise as a fully autologous and cost-effective biological scaffold for CTE

Conservative Treatment of Ewing’s Sarcoma of the Uterus in Young Women

Ewing sarcoma-primitive neuroectodermal tumors (ES/PNETs) constitute a family of neoplasms characterized by a continuum of neuroectodermal differentiations. ES/PNET of the uterus is rare. There are 48 cases of ES/PNET of the uterus published in the literature as far as we know. We describe a case of Ewing sarcoma of the uterus occurring in a 17-year-old woman presenting with a two-month history of pelvic pain. After surgical excision and microscopic, immunohistochemical, and electron microscopy examination, the diagnosis of Ewing sarcoma of the uterus was suggested. This report will discuss the diagnosis and surgical and clinical management of Ewing uterine sarcoma in young women, according to the available literature. In spite of the rarity of ES/PNETs, they should be taken into account in the differential diagnosis of uterine neoplasms in young women

Widespread retention of ohnologs in key developmental gene families following whole-genome duplication in arachnopulmonates

Whole-genome duplications (WGDs) have occurred multiple times during animal evolution, including in lineages leading to vertebrates, teleosts, horseshoe crabs, and arachnopulmonates. These dramatic events initially produce a wealth of new genetic material, generally followed by extensive gene loss. It appears, however, that developmental genes such as homeobox genes, signaling pathway components and microRNAs are frequently retained as duplicates (so-called ohnologs) following WGD. These not only provide the best evidence for WGD, but an opportunity to study its evolutionary consequences. Although these genes are well studied in the context of vertebrate WGD, similar comparisons across the extant arachnopulmonate orders are patchy. We sequenced embryonic transcriptomes from two spider species and two amblypygid species and surveyed three important gene families, Hox, Wnt, and frizzled, across these and 12 existing transcriptomic and genomic resources for chelicerates. We report extensive retention of putative ohnologs, further supporting the ancestral arachnopulmonate WGD. We also found evidence of consistent evolutionary trajectories in Hox and Wnt gene repertoires across three of the six arachnopulmonate orders, with interorder variation in the retention of specific paralogs. We identified variation between major clades in spiders and are better able to reconstruct the chronology of gene duplications and losses in spiders, amblypygids, and scorpions. These insights shed light on the evolution of the developmental toolkit in arachnopulmonates, highlight the importance of the comparative approach within lineages, and provide substantial new transcriptomic data for future study

Relevance of Positional Memory of Fibroblasts in Reprogramming to Induced Pluripotent Stem Cells

Induced Pluripotent Stem cells (iPSC) are adult skin fibroblasts (sFB) genetically reprogrammed to an embryonic stem cell-like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency.We hypothesize that the variability in the sFB reprogramming is due to the sFB used, as sFB derived from different anatomic sites exhibit topographic differentiation and preserve positional memory. Human sFB harvested from five different anatomical sites (neck, breast, arm, abdomen, thigh) were cultured for one week and their morphology, proliferation, and expression of mesenchymal or epithelial markers were evaluated by immunocytochemistry. Additionally, gene expression profile analysis was performed by real-time PCR including genes typically expressed in mesenchymal cells, and involved in cell growth, proliferation, development, andmorphogenesis. Intriguingly, while the morphology of sFB derived from different anatomical sites differed only slightly, proliferation rate and expression of distinctive markers varied greatly. Further, different sFB had different genetic program. Interestingly, sFB derived from neck and breast shared genetic signature of Mesenchymal Stem Cells, raising doubts about the existence of two distinct cell population. Since sFB topographic origin defines their genetic programitmight remarkably affect the efficiency of reprogramming. Hence, according to our evidence, it is mandatory to carefully select sFB population when planning sFB reprogramming for regenerative medicine purposes

From Cover to Core: Acellular Human Dermis for the Regeneration of Human Heart

Elasticity of myocardium is mostly due to elasticity of cardiomyocytes and is essential for cardiomyocyte alignment and differentiation. Cardiac decellularized ECM (d-ECM) is emerging as natural scaffold to promote and support myocardial regeneration. It is noteworthy that cardiac d-ECM is obtained through complete removal of cardiomyocytes with loss of elasticity. We hypothesize that decellularized skin might be an easily accessible, viable alternative for myocardium regeneration, as decellularization is unlikely to cause loss of skin elasticity, provided by elastic fibers rather than by resident cells. Skin fragments from patients undergoing plastic surgery were decellularized through novel simple and fast protocol. Decellularized Human Skin (d-HuSk) obtained was assayed in quantitative dye-binding method to measure content of elastin, while elastin distribution was evaluated on histological sections by Paraldehyde Fuchsin Gomori and Weigert Van Gieson stainings. d-Husk was then sectioned and used as scaffold to prepare three-dimensional culture of cardiac primitive cells (CPCs). Then, survival and ability of CPC cultured on d-HuSk to differentiate towards cardiac myocytes was evaluated at gene and protein level. Histological and quantitative analysis provided evidence of effective decellularization, preserved tissue architecture and retention of elastin. CPCs engrafted onto d-Husk, survived, and retained expression of markers specific for cardiac myocytes at gene and protein level.Our study provides compelling evidence that common signals act in cardiac and skin microenvironment to maintain CPC ability to differentiate towards cardiac muscle and that skin holds promise as an alternate biological scaffold for cardiovascular regenerative medicine

Decellularized human skin as biological scaffold for cardiovascular repair and regeneration

INTRODUCTION Skin shares properties of elasticity with muscular tissue. Since elasticity is mostly conferred by muscle cells or elastic fibers, after decellularization the removal of muscle cells causes in decellularized muscles loss of such property, while decellularized skin retains elasticity as skin ECM is rich in elastic fibers that are retained after decellularization. Additionally, mechanic properties are fundamental to ensure myocyte differentiation1 and alignment in myocardium. EXPERIMENTAL METHODS We developed a fast and efficient protocol of decellularization for human skin using skin fragments from patients undergoing plastic surgery. After decellularization, content of elastin was quantified by quantitative dye-binding method. Additionally elastin content and distribution was evaluated on histological sections by Paraldehyde Fuchsin Gomori and Weigert Van Gieson stainings. Decellularized Human SKin (d-HuSk) obtained was then sectioned into 600um thick sections and used as scaffold to prepare three-dimensional culture of cardiac primitive cells (CPCs). We evaluated, then, CPC survival and ability to differentiate, in vitro, towards cardiomyocytes at gene and protein level when cultured on d-HuSk. RESULTS AND DISCUSSION Decellularization procedure yielded the acellular extracellular matrix (ECM) with preserved tissue architecture, named d-HuSk. Importantly, histological and quantitative analysis clearly showed the retention of elastic fibers by d-HuSk. CPCs seeded on d-Husk engrafted and survived, and their ability to differentiate towards cardiomyocytes was not lost, as shown by preserved expression of markers specific for cardiac muscle cells, both at protein and gene level. Such results suggest that common signals and properties act both in cardiac and skin microenvironment, making skin a potential powerful and off-the-shelf biological scaffold for cardiovascular regenerative medicine. CONCLUSION Although emerging from an in vitro study, the evidence that progenitors of cardiac muscle lineage retain the ability to differentiate on biological scaffold obtained from different, more easily accessible, anatomic site, represents an important advance in cardiovascular regenerative medicine. Specifically, d-HuSk is an alternate biological scaffold that overcomes problems related to the preparation of myocardial biological scaffolds