The Microenvironment of Decellularized Extracellular Matrix from Heart Failure Myocardium Alters the Balance between Angiogenic and Fibrotic Signals from Stromal Primitive Cells.

Cardiac adverse remodeling is characterized by biological changes that affect the composition and architecture of the extracellular matrix (ECM). The consequently disrupted signaling can interfere with the balance between cardiogenic and pro-fibrotic phenotype of resident cardiac stromal primitive cells (CPCs). The latter are important players in cardiac homeostasis and can be exploited as therapeutic cells in regenerative medicine. Our aim was to compare the effects of human decellularized native ECM from normal (dECM-NH) or failing hearts (dECM-PH) on human CPCs. CPCs were cultured on dECM sections and characterized for gene expression, immunofluorescence, and paracrine profiles. When cultured on dECM-NH, CPCs significantly upregulated cardiac commitment markers (CX43, NKX2.5), cardioprotective cytokines (bFGF, HGF), and the angiogenesis mediator, NO. When seeded on dECM-PH, instead, CPCs upregulated pro-remodeling cytokines (IGF-2, PDGF-AA, TGF-ß) and the oxidative stress molecule H2O2. Interestingly, culture on dECM-PH was associated with impaired paracrine support to angiogenesis, and increased expression of the vascular endothelial growth factor (VEGF)-sequestering decoy isoform of the KDR/VEGFR2 receptor. Our results suggest that resident CPCs exposed to the pathological microenvironment of remodeling ECM partially lose their paracrine angiogenic properties and release more pro-fibrotic cytokines. These observations shed novel insights on the crosstalk between ECM and stromal CPCs, suggesting also a cautious use of non-healthy decellularized myocardium for cardiac tissue engineering approaches.

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.

Induced pluripotent stem cells (iPSCs) are adult somatic cells 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. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real-time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration-free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.

Prevalence of musculocutaneous nerve variations: Systematic review and meta-analysis.

We aimed to establish the prevalence of the musculocutaneous nerve (MCN) variations and the probability of the variation being pure or mixed in the same plexus. We applied the principles of evidence-based anatomy to find, appraise, and synthesize data through a meta-analysis of anatomical studies. The variations were grouped based on the presence and location of the communicating branch with the median nerve and the origin of branches to anterior arm muscles. Forty-three cadaveric studies met the inclusion criteria, providing data from 4124 plexuses. The overall pooled prevalence of plexuses with MCN variations was 20%. Based on the classification applied in our study, the pooled prevalence of variations was 17% in region 1A, 20% in region 1B, 36% in region 2 and 49% in region 3. Importantly, 64.58% of variations in region 1A and 74.14% of variations in region 1B were mixed, that is, associated with a variation in another region. The odds of finding another variation in the presence of a variation in region 2 or 3 were equal 0.37 and 0.52, respectively, demonstrating a significantly lower probability of finding mixed variations involving these regions, when compared with region 1A. Variations of the MCN are most common in the part distal to the exit from within or beneath the coracobrachialis muscle. Proximal variations are more often associated with another variation located along the nerve. These findings can assist health care professionals in the treatment of brachial plexus lesions. Clin. Anat. 32:183-195, 2019. © 2018 Wiley Periodicals, Inc.

Reinforcement of the pulmonary artery autograft with a polyglactin and polydioxanone mesh in the Ross operation: experimental study in growing lamb.

BACKGROUND AND AIM OF THE STUDY: Synthetic materials used for pulmonary autograft (PA) reinforcement in the Ross procedure fail to match the demand for structural growth, have only a limited longevity, and do not guarantee adequate vascular compliance in high-pressure load systems. The study aim was to develop a resorbable reinforcement of a PA, tailored to provide structural support and to guide the process of wall structure modification for the preservation of graft viability. METHODS: An experimental model of translocation of the pulmonary trunk as an autograft in the aortic position was developed and performed under cardiopulmonary bypass in young lambs. The PA was left without reinforcement, reinforced with standard commercially available mesh, or reinforced with resorbable mesh of polyglactin and polydioxanone. RESULTS: Based on vessel diameter measurements by transesophageal echography at day 0 and at six months postoperatively, only the PA with resorbable reinforcement showed a behavior similar to that of the normal aorta in a growing lamb. With the non-resorbable reinforcement, transmural migration of the mesh was observed, accompanied by a conspicuous inflammatory infiltrate and fibrosis. CONCLUSION: The resorbable mesh allowed for histological wall modification, characterized by the presence of highly organized smooth muscle cells and elastic lamellae in the media. The mechanical and histological features of this resorbable mesh-reinforced PA may be crucial to the clinical long-term success of the Ross procedure.

Decellularized Extracellular Matrix for Modeling Cardiac Extracellular Microenvironment.

The extracellular matrix (ECM) forms most of the tissue microenvironment and is in a constant and dynamic equilibrium with cells. The decellularization process employs physical or chemical methods, or a combination of them, to remove the cellular components of tissues and organs while preserving the architecture and composition of the ECM. Depending on the methodology used, the decellularized ECM (dECM) is then suitable for research or clinical applications. Here, we describe an optimized protocol for the efficient decellularization of the human myocardium to generate 3D scaffolds of well-preserved cardiac extracellular matrix that can be used for in vitro or in vivo studies.

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating.

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

Cardiac primitive cells become committed to a cardiac fate in adult human heart with chronic ischemic disease but fail to acquire mature phenotype: genetic and phenotypic study.

Adult human heart hosts a population of cardiac primitive CD117-positive cells (CPCs), which are responsible for physiological tissue homeostasis and regeneration. While the bona fide stem cells express telomerase, their progenies are no longer able to preserve telomeric DNA; hence the balance between their proliferation and differentiation has to be tightly controlled in order to prevent cellular senescence and apoptosis of CPCs before their maturation can be accomplished. We have examined at cellular and molecular level the proliferation, apoptosis and commitment of CPCs isolated from normal (CPC-N) and age-matched pathological adult human hearts (CPC-P) with ischemic heart disease. In the CPC-P, genes related to early stages of developmental processes, nervous system development and neurogenesis, skeletal development, bone and cartilage development were downregulated, while those involved in mesenchymal cell differentiation and heart development were upregulated, together with the transcriptional activation of TGFß/BMP signaling pathway. In the pathological heart, asymmetric division was the prevalent type of cardiac stem cell division. The population of CPC-P consisted mainly of progenitors of cardiac cell lineages and less precursors; these cells proliferated more, but were also more susceptible to apoptosis with respect to CPC-N. These results indicate that CPCs fail to reach terminal differentiation and functional competence in pathological conditions. Adverse effects of underlying pathology, which disrupts cardiac tissue structure and composition, and cellular senescence, resulting from cardiac stem cell activation in telomere dysfunctional environment, can be responsible for such outcome.

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype¼, while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Cardiac shock wave therapy: assessment of safety and new insights into mechanisms of tissue regeneration.

Although low-energy extracorporeal cardiac shock wave (ECSW) therapy represents an attractive non-invasive treatment option for ischaemic heart disease, the precise mechanisms of its action and influence on the cardiac tissue remain obscure. The goal of this study was to evaluate the effects of SW application on cardiac function and structure. Four-month-old Fisher 344 rats were subjected to ECSW therapy. Echocardiographic measurements of cardiac function were performed at baseline and at 1 and 3 months after treatment. Signs of inflammation, apoptosis and fibrosis were evaluated by immunohistochemistry in the control and treated hearts. ECSW application did not provoke arrhythmia or increase the troponin-I level. At all time points, the left ventricular ejection fraction and fractional shortening remained stable. Histological analysis revealed neither differences in the extracellular matrix collagen content nor the presence of fibrosis; similarly, there were no signs of inflammation. Moreover, a population of cardiac cells that responded eagerly to ECSW application in the adult heart was identified; c-kit-positive, Ki67-positive, orthochromatic cells, corresponding to cardiac primitive cells, were 2.65-fold more numerous in the treated myocardium. In conclusion, non-invasive ECSW therapy is a safe and effective way of activating cardiac stem cells and myocardial regeneration. Because many factors influence cellular turnover in the ischaemic myocardium during the course of ischaemic heart disease, cardiac remodelling, and heart failure progression, studies to identify the optimal treatment time are warranted.

Non-modified RNA-Based Reprogramming of Human Dermal Fibroblasts into Induced Pluripotent Stem Cells.

The generation of pluripotent stem cells from adult somatic cells by cell reprogramming has put a whole new perspective on stem cell biology and stem cell-based regenerative medicine. Cell reprogramming acts through the introduction of key genes that regulate and maintain the pluripotent cell state. In this chapter, we describe the optimized protocol for the efficient isolation of fibroblasts from a skin punch biopsy and the subsequent easy and effective generation of integration-free induced pluripotent stem cell (iPSC) colonies forcing the expression of specific factors by non-modified RNAs.

Cardiac-derived extracellular matrix: A decellularization protocol for heart regeneration.

Extracellular matrix (ECM) is a fundamental component of the heart, guiding vital cellular processes during organ homeostasis. Most cardiovascular diseases lead to a remarkable remodeling of the ECM, accompanied by the formation of a fibrotic tissue that heavily compromises the heart function. Effective therapies for managing fibrosis and promoting physiological ECM repair are not yet available. The production of a decellularized extracellular matrix (d-ECM) serving as a three-dimensional and bioactive scaffold able to modulate cellular behavior and activities is considered crucial to achieve a successful regeneration. The protocol represents a step-by-step method to obtain a decellularized cardiac matrix through the combination of sodium dodecyl sulphate (SDS) and Triton X-100. Briefly, cardiac samples obtained from left ventricles of explanted, pathological human hearts were dissected and washed to remove residual body fluids. Samples were then snap-frozen and sliced by a cryostat into 350 µm thick sections. The sections obtained were decellularized using a solution containing 1% Triton X-100 and 1% SDS in combination, for 24 hours, until observing the color change from brownish-red to translucent-white. As a result, the protocol shows efficiency in preserving ECM architecture and protein composition during the whole process, suggesting that it is worthwhile, highly reproducible and produces a well- preserved decellularized extracellular matrix from cardiac samples. Notwithstanding, some limitations need to be addressed, such as the risk for microbial contamination and the unpredictable trend of the protocol when applied to decellularize samples other than myocardium, vessels, or skin. These issues require antibiotics mixture supplement during the procedure followed by UV sterilization, and appropriate adjustments for a tissue-specific utilization, respectively. The protocol is intended to produce a cardiac d-ECM for cell settlement, representing the ideal scaffold for tissue engineering purposes.

Human Cardiac Progenitor Cell-Derived Extracellular Vesicles Exhibit Promising Potential for Supporting Cardiac Repair in Vitro.

Although human Cardiac Progenitor Cells (hCPCs) are not retained by host myocardium they still improve cardiac function when injected into ischemic heart. Emerging evidence supports the hypothesis that hCPC beneficial effects are induced by paracrine action on resident cells. Extracellular vesicles (EVs) are an intriguing mechanism of cell communication based on the transport and transfer of peptides, lipids, and nucleic acids that have the potential to modulate signaling pathways, cell growth, migration, and proliferation of recipient cells. We hypothesize that EVs are involved in the paracrine effects elicited by hCPCs and held accountable for the response of the infarcted myocardium to hCPC-based cell therapy. To test this theory, we collected EVs released by hCPCs isolated from healthy myocardium and evaluated the effects they elicited when administered to resident hCPC and cardiac fibroblasts (CFs) isolated from patients with post-ischemic end-stage heart failure. Evidence emerging from our study indicated that hCPC-derived EVs impacted upon proliferation and survival of hCPCs residing in the ischemic heart and regulated the synthesis and deposition of extracellular-matrix by CFs. These findings suggest that beneficial effects exerted by hCPC injection are, at least to some extent, ascribable to the delivery of signals conveyed by EVs.

Biomimetic design of bioartificial scaffolds for the in vitro modelling of human cardiac fibrosis.

In vitro models of pathological cardiac tissue have attracted interest as predictive platforms for preclinical validation of therapies. However, models reproducing specific pathological features, such as cardiac fibrosis size (i.e., thickness and width) and stage of development are missing. This research was aimed at engineering 2D and 3D models of early-stage post-infarct fibrotic tissue (i.e., characterized by non-aligned tissue organization) on bioartificial scaffolds with biomimetic composition, design, and surface stiffness. 2D scaffolds with random nanofibrous structure and 3D scaffolds with 150 µm square-meshed architecture were fabricated from polycaprolactone, surface-grafted with gelatin by mussel-inspired approach and coated with cardiac extracellular matrix (ECM) by 3 weeks culture of human cardiac fibroblasts. Scaffold physicochemical properties were thoroughly investigated. AFM analysis of scaffolds in wet state, before cell culture, confirmed their close surface stiffness to human cardiac fibrotic tissue. Following 3 weeks culture, biomimetic biophysical and biochemical scaffold properties triggered the activation of myofibroblast phenotype. Upon decellularization, immunostaining, SEM and two-photon excitation fluorescence microscopy showed homogeneous decoration of both 2D and 3D scaffolds with cardiac ECM. The versatility of the approach was demonstrated by culturing ventricular or atrial cardiac fibroblasts on scaffolds, thus suggesting the possibility to use the same scaffold platforms to model both ventricular and atrial cardiac fibrosis. In the future, herein developed in vitro models of cardiac fibrotic tissue, reproducing specific pathological features, will be exploited for a fine preclinical tuning of therapies.

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Patent Foramen Ovale-A Not So Innocuous Septal Atrial Defect in Adults.

Patent foramen ovale (PFO) is a common congenital atrial septal defect with an incidence of 15-35% in the adult population. The development of the interatrial septum is a process that begins in the fourth gestational week and is completed only after birth. During intrauterine life, the foramen ovale allows the passage of highly oxygenated blood from the right to the left atrium and into the systemic arteries, thus bypassing the pulmonary circulation. In 75% of the general population, the foramen ovale closes after birth, and only an oval depression, called fossa ovalis, remains on the right side of the interatrial septum. Patent foramen ovale can be associated with various clinically important conditions, including migraine and stroke, or decompression illness in divers. The aim of this review is to summarize the PFO developmental and anatomical features and to discuss the clinical risks associated with this atrial septal defect in adults.

Correlation between Official and Common Field-Based Fitness Tests in Elite Soccer Referees.

Official tests are used to assess the fitness status of soccer referees, and their results correlate with match performance. However, FIFA-approved tests expose the referees to high physical demands and are difficult to implement during the sportive year. The aim of our study was to evaluate the correlation between the 6 × 40-m sprint and Yo-Yo Intermittent Recovery Level 1 (IR1) official tests and other field-based tests that require no or little equipment, are not time-consuming, and impose low physical demands. All tests were performed by male referees from the Regional Section of the Italian Referee Association (n = 30). We observed a strong correlation between 6 × 40-m sprint and Illinois agility tests (r = 0.63, p = 0.001) and a moderate correlation between Yo-Yo IR1 and hand-grip strength in the dominant (r = 0.45, p = 0.014) and non-dominant hand (r = 0.41, p = 0.031). Interestingly, only a moderate correlation (r = -0.42, p = 0.025) was observed between the FIFA official tests, 6 × 40-m sprint and Yo-Yo IR1. These results suggest that Illinois agility and hand-grip tests could represent simple and low-physical-impact tools for repeated assessment and monitoring of referee fitness throughout the sportive season.

Epithelial-mesenchymal transition of epicardial mesothelium is a source of cardiac CD117-positive stem cells in adult human heart.

Epithelial-mesenchymal transition is implicated in the remodelling of tissues during development and in the adult life. In the heart, it gives origin to progenitors of fibroblasts, coronary endothelium, smooth muscle cells, and cardiomyocytes. Moreover, epicardially-derived cells determine myocardial wall thickness and Purkinje fibre network. Recently, the presence of numerous cardiac stem cells in the subepicardium of the adult human heart has been described and the hypothesis that epicardially-derived cells can contribute to the population of cardiac stem cells in the adult heart has been advanced. In an effort to test this hypothesis and establish a possible link between epicardium, epicardially-derived cells and cardiac stem cells in the adult human heart we have examined epicardial mesothelial cells in the normal and pathological adult human heart with ischemic cardiomyopathy in vivo and we have induced and documented their epithelial-mesenchymal transition in vitro. Noticeably, epicardial cells were missing from the surface of pathological hearts and the cells with the expression of epithelial and mesenchymal markers populated thick subepicardial space. When the fragments of epicardium from the normal hearts were cultured on the specific substrate formed by extracellular matrix derived from cardiac fibroblasts, we obtained the outgrowth of the epithelial sheet with the mRNA and protein expression characteristic of epicardium. TGFß induced cellular and molecular changes typical of epithelial-mesenchymal transition. Moreover, the epicardially-derived cells expressed CD117 antigen. Thus, this study provides evidence that cardiac stem cells can originate from epithelial-mesenchymal transition of the epicardial cells in the adult human heart.

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Compact and tunable stretch bioreactor advancing tissue engineering implementation. Application to engineered cardiac constructs.

Physical stimuli are crucial for the structural and functional maturation of tissues both in vivo and in vitro. In tissue engineering applications, bioreactors have become fundamental and effective tools for providing biomimetic culture conditions that recapitulate the native physical stimuli. In addition, bioreactors play a key role in assuring strict control, automation, and standardization in the production process of cell-based products for future clinical application. In this study, a compact, easy-to-use, tunable stretch bioreactor is proposed. Based on customizable and low-cost technological solutions, the bioreactor was designed for providing tunable mechanical stretch for biomimetic dynamic culture of different engineered tissues. In-house validation tests demonstrated the accuracy and repeatability of the imposed mechanical stimulation. Proof of concepts biological tests performed on engineered cardiac constructs, based on decellularized human skin scaffolds seeded with human cardiac progenitor cells, confirmed the bioreactor Good Laboratory Practice compliance and ease of use, and the effectiveness of the delivered cyclic stretch stimulation on the cardiac construct maturation.

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.