Forward programming of hiPSCs towards beta-like cells using Ngn3, Pdx1, and MafA.

Transplantation of stem cell-derived ß-cells is a promising therapeutic advancement in the treatment of type 1 diabetes mellitus. A current limitation of this approach is the long differentiation timeline that generates a heterogeneous population of pancreatic endocrine cells. To address this limitation, an inducible lentiviral overexpression system of mature ß-cell markers was introduced into human induced-pluripotent stem cells (hiPSCs). Following the selection of the successfully transduced hiPSCs, the cells were treated with doxycycline in the pancreatic progenitor induction medium to support their transition toward the pancreatic lineage. Cells cultured with doxycycline presented the markers of interest, NGN3, PDX1, and MAFA, after five days of culture, and glucose-stimulated insulin secretion assays demonstrated that the cells were glucose-responsive in a monolayer culture. When cultured as a spheroid, the markers of interest and insulin secretion in a static glucose-stimulated insulin secretion assay were maintained; however, insulin secretion upon consecutive glucose challenges was limited. Comparison to human fetal and adult donor tissues identified that although the hiPSC-derived spheroids present similar markers to adult insulin-producing cells, they are functionally representative of fetal development. Together, these results suggest that with optimization of the temporal expression of these markers, forward programming of hiPSCs towards insulin-producing cells could be a possible alternative for islet transplantation.

Exploring the relationship between epigenetic DNA methylation and cardiac fibrosis through Raman microspectroscopy.

Cardiomyopathies are associated with fibrotic remodeling of the heart, which is characterized by the excessive accumulation of collagen type I (COL I) due to chronic inflammation and suspected epigenetic influences. Despite the severity and high mortality rate of cardiac fibrosis, current treatment options are often inadequate, underscoring the importance of gaining a deeper understanding of the disease's underlying molecular and cellular mechanisms. In this study, the extracellular matrix (ECM) and nuclei in fibrotic areas of different cardiomyopathies were molecularly characterized by Raman microspectroscopy and imaging and compared with the control myocardium. Patient samples were obtained from heart tissue affected by ischemia, hypertrophy, and dilated cardiomyopathy and analyzed for fibrosis through conventional histology and marker-independent Raman microspectroscopy (RMS). Prominent differences between control myocardium and cardiomyopathies were revealed by spectral deconvolution of COL I Raman spectra. Statistically significant differences were identified in the amide I region of spectral subpeak at 1,608 cm-1, which is a representative endogenous marker for alterations in the structural conformation of COL I fibers. Moreover, epigenetic 5mC DNA modification was identified within cell nuclei by multivariate analysis. A statistically significant increase in signal intensities of spectral features indicative of DNA methylation was detected in cardiomyopathies in accordance with immunofluorescence 5mC staining. Overall, RMS is a versatile technology in the discrimination of cardiomyopathies based on molecular evaluation of COL I and nuclei while providing insights into the pathogenesis of the diseases.NEW & NOTEWORTHY Cardiomyopathies are associated with severe fibrotic remodeling of the heart, which is characterized by the excessive accumulation of collagen type I (COL I). In this study, we used marker-independent Raman microspectroscopy (RMS) to gain a deeper understanding of the disease's underlying molecular and cellular mechanisms.

Monitoring the macrophage response towards biomaterial implants using label-free imaging.

Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation.

Raman microspectroscopy identifies fibrotic tissues in collagen-related disorders via deconvoluted collagen type I spectra.

Fibrosis is a consequence of the pathological remodeling of extracellular matrix (ECM) structures in the connective tissue of an organ. It is often caused by chronic inflammation, which over time, progressively leads to an excess deposition of collagen type I (COL I) that replaces healthy tissue structures, in many cases leaving a stiff scar. Increasing fibrosis can lead to organ failure and death; therefore, developing methods that potentially allow real-time monitoring of early onset or progression of fibrosis are highly valuable. In this study, the ECM structures of diseased and healthy human tissue from multiple organs were investigated for the presence of fibrosis using routine histology and marker-independent Raman microspectroscopy and Raman imaging. Spectral deconvolution of COL I Raman spectra allowed the discrimination of fibrotic and non-fibrotic COL I fibers. Statistically significant differences were identified in the amide I region of the spectral subpeak at 1608 cm-1, which was deemed to be representative for structural changes in COL I fibers in all examined fibrotic tissues. Raman spectroscopy-based methods in combination with this newly discovered spectroscopic biomarker potentially offer a diagnostic approach to non-invasively track and monitor the progression of fibrosis. STATEMENT OF SIGNIFICANCE: Current diagnosis of fibrosis still relies on histopathological examination with invasive biopsy procedures. Although, several non-invasive imaging techniques such as positron emission tomography, single-photon emission computed tomography and second harmonic generation are gradually employed in preclinical or clinical studies, these techniques are limited in spatial resolution and the morphological interpretation highly relies on individual experience and knowledge. In this study, we propose a non-destructive technique, Raman microspectroscopy, to discriminate fibrotic changes of collagen type I based on a molecular biomarker. The changes of the secondary structure of collagen type I can be identified by spectral deconvolution, which potentially can provide an automatic diagnosis for fibrotic tissues in the clinical applicaion.

Decorin improves human pancreatic ß-cell function and regulates ECM expression in vitro.

Transplantation of islets of Langerhans is a promising alternative treatment strategy in severe cases of type 1 diabetes mellitus; however, the success rate is limited by the survival rate of the cells post-transplantation. Restoration of the native pancreatic niche during transplantation potentially can help to improve cell viability and function. Here, we assessed for the first time the regulatory role of the small leucine-rich proteoglycan decorin (DCN) in insulin secretion in human ß-cells, and its impact on pancreatic extracellular matrix (ECM) protein expression in vitro. In depth analyses utilizing next-generation sequencing as well as Raman microspectroscopy and Raman imaging identified pathways related to glucose metabolism to be upregulated in DCN-treated cells, including oxidative phosphorylation within the mitochondria as well as proteins and lipids of the endoplasmic reticulum. We further showed the effectiveness of DCN in a transplantation setting by treating collagen type 1-encapsulated ß-cell-containing pseudo-islets with DCN. Taken together, in this study, we demonstrate the potential of DCN to improve the function of insulin-secreting ß-cells while reducing the expression of ECM proteins affiliated with fibrotic capsule formation, making DCN a highly promising therapeutic agent for islet transplantation.

Type 1 diabetes and engineering enhanced islet transplantation.

The development of new therapeutic approaches to treat type 1 diabetes mellitus (T1D) relies on the precise understanding and deciphering of insulin-secreting ß-cell biology, as well as the mechanisms responsible for their autoimmune destruction. ß-cell or islet transplantation is viewed as a potential long-term therapy for the millions of patients with diabetes. To advance the field of insulin-secreting cell transplantation, two main research areas are currently investigated by the scientific community: (1) the identification of the developmental pathways that drive the differentiation of stem cells into insulin-producing cells, providing an inexhaustible source of cells; and (2) transplantation strategies and engineered transplants to provide protection and enhance the functionality of transplanted cells. In this review, we discuss the biology of pancreatic ß-cells, pathology of T1D and current state of ß-cell differentiation. We give a comprehensive view and discuss the different possibilities to engineer enhanced insulin-secreting cell/islet transplantation from a translational perspective.

Noninvasive Physical Plasma as Innovative and Tissue-Preserving Therapy for Women Positive for Cervical Intraepithelial Neoplasia.

(1) Background: Cervical intraepithelial neoplasia (CIN) of long-term persistence or associated with individual treatment indications often requires highly invasive treatments. These are associated with risks of bleeding, infertility, and pregnancy complications. For low- and middle-income countries (LMICs), standard treatment procedures are difficult to implement and manage. We characterized the application of the highly energized gas "noninvasive physical plasma" (NIPP) for tissue devitalization and the treatment of CIN. (2) Methods: We report the establishment of a promising tissue devitalization procedure by NIPP application. The procedure was characterized at the in vitro, ex vivo and in vivo levels. We performed the first prospective, single-armed phase-IIb trial in 20 CIN1/2 patients (NCT03218436). (3) Results: NIPP-treated cervical cancer cells used as dysplastic in vitro model exhibited significant cell growth retardation due to DNA damage, cell cycle arrest and apoptosis. Ex vivo and in vivo tissue assessments showed a highly noninvasive and tissue-preserving treatment procedure which induces transmucosal tissue devitalization. Twenty participants were treated with NIPP and attended a 24-week follow-up. Treatment success was achieved in 19 (95%) participants without postinterventional complications other than mild to moderate discomfort during application. (4) Conclusions: The results from this study preliminarily suggest that NIPP could be used for an effective and tissue-preserving treatment for CIN without the disadvantages of standard treatments. However, randomized controlled trials must confirm the efficacy and noninferiority of NIPP compared to standard treatments.

Basement membrane proteins improve human islet survival in hypoxia: Implications for islet inflammation.

Enzymatic digestion of the pancreas during islet isolation is associated with disintegration of the islet basement membrane (IBM) that can cause reduction of functional and morphological islet integrity. Attempts to re-establish IBM by coating the surface of culture vessels with various IBM proteins (IBMP) have resulted in loss of islet phenotype and function. This study investigated the capability of Collagen-IV, Laminin-521 and Nidogen-1, utilised as single or combined media supplements, to protect human islets cultured in hypoxia. When individually supplemented to media, all IBMP significantly improved islet survival and in-vitro function, finally resulting in as much as a two-fold increase of islet overall survival. In contrast, combining IBMP enhanced the production of chemokines and reactive oxygen species diminishing all positive effects of individually added IBMP. This impact was concentration-dependent and concerned nearly all parameters of islet integrity. Predictive extrapolation of these findings to data from 116 processed human pancreases suggests that more than 90% of suboptimal pancreases could be rescued for clinical islet transplantation increasing the number of transplantable preparations from actual 25 to 40 when adding Nidogen-1 to pretransplant culture. This study suggests that media supplementation with essential IBMP protects human islets from hypoxia. Amongst those, certain IBMP may be incompatible when combined or applied at higher concentrations. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally-invasive treatment that can reverse type 1 diabetes in certain patients. It involves infusing of insulin-producing cell-clusters (islets) from donor pancreases. Unfortunately, islet extraction is associated with damage of the islet basement membrane (IBM) causing reduced islet function and cell death. Attempts to re-establish the IBM by coating the surface of culture vessels with IBM proteins (IBMP) have been unsuccessful. Instead, we dissolved the most relevant IBM components Collagen-IV, Laminin-521 and Nidogen-1 in media routinely used for clinical islet culture and transplantation. We found human islet survival and function was substantially improved by IBMP, particularly Nidogen-1, when exposed to a hypoxic environment as found in vivo. We also investigated IBMP combinations. Our present findings have important clinical implications.

Raman Imaging and Fluorescence Lifetime Imaging Microscopy for Diagnosis of Cancer State and Metabolic Monitoring.

Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

Hyaluronic Acid-Functionalized Hybrid Gelatin-Poly-L-Lactide Scaffolds with Tunable Hydrophilicity.

In this study, we describe the production of hybrid gelatin-poly-L-lactide electrospun scaffolds whose hydrophilicity was controlled by binding increasing concentrations of hyaluronic acid (HA). We show that cross-linking has advantages over coating when aiming to functionalize the scaffolds with HA. The here described scaffolds structurely mimicked the complexity of the extracellular matrix, and when excited by second harmonic generation, they produced a signal that is typical of collagen-containing biological fibers. Fluorescence lifetime imaging microscopy (FLIM) was used to marker-independently monitor the growth of human dermal fibroblasts on the electrospun scaffolds using reduced (phosphorylated) nicotinamide adenine dinucleotide as target. Benefitting from the different fluorescence lifetimes of the polymer and the endogenous cellular fluorophore, we were able to distinguish and separate the signals produced by the cells from the signals generated by the electrospun scaffolds. FLIM further allowed the detection of metabolic differences in the cells seeded on the HA-functionalized scaffolds compared with cells that were cultured on nonfunctionalized control scaffolds.

The Foreign Body Response to an Implantable Therapeutic Reservoir in a Diabetic Rodent Model.

Advancements in type 1 diabetes mellitus treatments have vastly improved in recent years. The move toward a bioartificial pancreas and other fully implantable systems could help restore patient's glycemic control. However, the long-term success of implantable medical devices is often hindered by the foreign body response. Fibrous encapsulation "walls off" the implant to the surrounding tissue, impairing its functionality. In this study we aim to examine how streptozotocin-induced diabetes affects fibrous capsule formation and composition surrounding implantable drug delivery devices following subcutaneous implantation in a rodent model. After 2 weeks of implantation, the fibrous capsule surrounding the devices were examined by means of Raman spectroscopy, micro-computed tomography (µCT), and histological analysis. Results revealed no change in mean fibrotic capsule thickness between diabetic and healthy animals as measured by µCT. Macrophage numbers (CCR7 and CD163 positive) remained similar across all groups. True component analysis also showed no quantitative difference in the alpha-smooth muscle actin and extracellular matrix proteins. Although principal component analysis revealed significant secondary structural difference in collagen I in the diabetic group, no evidence indicates an influence on fibrous capsule composition surrounding the device. This study confirms that diabetes did not have an effect on the fibrous capsule thickness or composition surrounding our implantable drug delivery device. Impact Statement Understanding the impact diabetes has on the foreign body response (FBR) to our implanted material is essential for developing an effective drug delivery device. We used several approaches (Raman spectroscopy and micro-computed tomography imaging) to demonstrate a well-rounded understanding of the diabetic impact on the FBR to our devices, which is imperative for its clinical translation.

Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration.

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet ß-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic ß-cells via the αvß3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.

Collagen and Endothelial Cell Coculture Improves ß-Cell Functionality and Rescues Pancreatic Extracellular Matrix.

The use of biomaterials and biomaterial functionalization is a promising approach to support pancreatic islet viability posttransplantation in an effort to reduce insulin dependence for patients afflicted with diabetes mellitus type 1. Extracellular matrix (ECM) proteins are known to impact numerous reparative functions in the body. Assessing how endogenously expressed pancreatic ECM proteins are affected by posttransplant-like hypoxic conditions may provide significant insights toward the development of tissue-engineered therapeutic strategies to positively influence ß-cell survival, proliferation, and functionality. Here, we investigated the expression of three relevant groups of pancreatic ECM proteins in human native tissue, including basement membrane (BM) proteins (collagen type 4 [COL4], laminins [LAM]), proteoglycans (decorin [DCN], nidogen-1 [NID1]), and fibril-forming proteins (fibronectin [FN], collagen type 1 [COL1]). In an in vitro hypoxia model, we identified that ECM proteins were differently affected by hypoxic conditions, contributing to an overall loss of ß-cell functionality. The use of a COL1 hydrogel as carrier material demonstrated a protective effect on ß-cells mitigating the effect of hypoxia on proteoglycans as well as fibril-forming protein expression, supporting ß-cell functionality in hypoxia. We further showed that providing endothelial cells (ECs) into the COL1 hydrogel improves ß-cell response as well as the expression of relevant BM proteins. Our data show that ß-cells benefit from a microenvironment composed of structure-providing COL1 with the incorporation of ECs to withstand the harsh conditions of hypoxia. Such hydrogels support ß-cell survival and can serve as an initial source of ECM proteins to allow cell engraftment while preserving cell functionality posttransplantation. Impact statement Expression analysis identifies hypoxia-induced pathological changes in extracellular matrix (ECM) homeostasis as potential targets to support ß-cell transplants by encapsulation in biomaterials for the treatment of diabetes mellitus. A collagen-1 hydrogel is shown to attenuate the effect of hypoxia on ß-cells and their ECM expression. The functionalization of the hydrogel with endothelial cells increases the ß-cell response to glucose and rescues essential basement membrane proteins.

Fluorescence lifetime metabolic mapping of hypoxia-induced damage in pancreatic pseudo-islets.

Pancreatic islet isolation from donor pancreases is an essential step for the transplantation of insulin-secreting ß-cells as a therapy to treat type 1 diabetes mellitus. This process however damages islet basement membranes, which can lead to islet dysfunction or death. Posttransplantation, islets are further stressed by a hypoxic environment and immune reactions that cause poor engraftment and graft failure. The current standards to assess islet quality before transplantation are destructive procedures, performed on a small islet population that does not reflect the heterogeneity of large isolated islet batches. In this study, we incorporated fluorescence lifetime imaging microscopy (FLIM) into a pancreas-on-chip system to establish a protocol to noninvasively assess the viability and functionality of pancreatic ß-cells in a three-dimensional in vitro model (= pseudo-islets). We demonstrate how (pre-) hypoxic ß-cell-composed pseudo-islets can be discriminated from healthy functional pseudo-islets according to their FLIM-based metabolic profiles. The use of FLIM during the pretransplantation pancreatic islet selection process has the potential to improve the outcome of ß-cell islet transplantation.

Trans-Mucosal Efficacy of Non-Thermal Plasma Treatment on Cervical Cancer Tissue and Human Cervix Uteri by a Next Generation Electrosurgical Argon Plasma Device.

Non-invasive physical plasma (NIPP) generated by non-thermally operated electrosurgical argon plasma sources is a promising treatment for local chronic inflammatory, precancerous and cancerous diseases. NIPP-enabling plasma sources are highly available and medically approved. The purpose of this study is the investigation of the effects of non-thermal NIPP on cancer cell proliferation, viability and apoptosis and the identification of the underlying biochemical and molecular modes of action. For this, cervical cancer (CC) single cells and healthy human cervical tissue were analyzed by cell counting, caspase activity assays, microscopic and flow-cytometric viability measurements and molecular tissue characterization using Raman imaging. NIPP treatment caused an immediate and persisting decrease in CC cell growth and cell viability associated with significant plasma-dependent effects on lipid structures. These effects could also be identified in primary cells from healthy cervical tissue and could be traced into the basal cell layer of superficially NIPP-treated cervical mucosa. This study shows that NIPP treatment with non-thermally operated electrosurgical argon plasma devices is a promising method for the treatment of human mucosa, inducing specific molecular changes in cells.

Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model.

The increasing prevalence of diabetes, its heterogeneity, and the limited number of treatment options drive the need for physiologically relevant assay platforms with human genetic background that have the potential to improve mechanistic understanding and e\xpedite diabetes-related research and treatment. In this study, we developed an endocrine pancreas-on-a-chip model based on a tailored microfluidic platform, which enables self-guided trapping of single human pseudo-islets. Continuous, low-shear perfusion provides a physiologically relevant microenvironment especially important for modeling and monitoring of the endocrine function as well as sufficient supply with nutrients and oxygen. Human pseudo-islets, generated from the conditionally immortalized EndoC-ßH3 cell line, were successfully injected by hydrostatic pressure-driven flow without altered viability. To track insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, dynamic sampling of the supernatant as well as non-invasive real-time monitoring using Raman microspectroscopy was established on-chip. Dynamic sampling indicated a biphasic glucose-stimulated insulin response. Raman microspectroscopy allowed to trace glucose responsiveness in situ and to visualize different molecular structures such as lipids, mitochondria and nuclei. In-depth spectral analyses demonstrated a glucose stimulation-dependent, increased mitochondrial activity, and a switch in lipid composition of insulin secreting vesicles, supporting the high performance of our pancreas-on-a-chip model.

The role of extracellular matrix in biomechanics and its impact on bioengineering of cells and 3D tissues.

The cells and tissues of the human body are constantly exposed to exogenous and endogenous forces that are referred to as biomechanical cues. They guide and impact cellular processes and cell fate decisions on the nano-, micro- and macro-scale, and are therefore critical for normal tissue development and maintaining tissue homeostasis. Alterations in the extracellular matrix composition of a tissue combined with abnormal mechanosensing and mechanotransduction can aberrantly activate signaling pathways that promote disease development. Such processes are therefore highly relevant for disease modelling or when aiming for the development of novel therapies. In this mini review, we describe the main biomechanical cues that impact cellular fates. We highlight their role during development, homeostasis and in disease. We also discuss current techniques and tools that allow us to study the impact of biomechanical cues on cell and tissue development under physiological conditions, and we point out directions, in which in vitro biomechanics can be of use in the future.

Controlled Heterotypic Pseudo-Islet Assembly of Human ß-Cells and Human Umbilical Vein Endothelial Cells Using Magnetic Levitation.

ß-Cell functionality and survival are highly dependent on the cells' microenvironment and cell-cell interactions. Since the pancreas is a highly vascularized organ, the crosstalk between ß-cells and endothelial cells (ECs) is vital to ensure proper function. To understand the interaction of pancreatic ß-cells with vascular ECs, we sought to investigate the impact of the spatial distribution on the interaction of human cell line-based ß-cells (EndoC-ßH3) and human umbilical vein endothelial cells (HUVECs). We focused on the evaluation of three major spatial distributions, which can be found within human islets in vivo, in tissue-engineered heterotypic cell spheroids, so-called pseudo-islets, by controlling the aggregation process using magnetic levitation. We report that heterotypic spheroids formed by spontaneous aggregation cannot be maintained in culture due to HUVEC disassembly over time. In contrast, magnetic levitation allows the formation of stable heterotypic spheroids with defined spatial distribution and significantly facilitated HUVEC integration. To the best of our knowledge, this is the first study that introduces a human-only cell line-based in vitro test system composed of a coculture of ß-cells and ECs with a successful stimulation of ß-cell secretory function monitored by a glucose-stimulated insulin secretion assays. In addition, we systematically investigate the impact of the spatial distribution on cocultures of human ß-cells and ECs, showing that the architecture of pseudo-islets significantly affects ß-cell functionality. Impact statement Tissue engineering of coculture systems containing ß-cells and endothelial cells (ECs) is a promising technique to stimulate ß-cell functionality. In this study, we analyzed human pancreatic islet tissue and revealed three different native distributions of ß-cells and ECs. We successfully recreated these distributions in vitro by employing magnetic levitation of human ß-cells and ECs, forming controlled heterotypic pseudo-islets, which enabled us to identify a significant impact of the pseudo-islet architecture on insulin secretion.

Improved long-term durability of allogeneic heart valves in the orthotopic sheep model.

OBJECTIVES: Frozen cryopreservation (FC) with the vapour phase of liquid nitrogen storage (-135°C) is a standard biobank technique to preserve allogeneic heart valves to enable a preferable allograft valve replacement in clinical settings. However, their long-term function is limited by immune responses, inflammation and structural degeneration. Ice-free cryopreserved (IFC) valves with warmer storage possibilities at -80°C showed better matrix preservation and decreased immunological response in preliminary short-term in vivo studies. Our study aimed to assess the prolonged performance of IFC allografts in an orthotopic pulmonary sheep model. METHODS: FC (n = 6) and IFC (n = 6) allografts were transplanted into juvenile Merino sheep. After 12 months of implantation, functionality testing via 2-dimensional echocardiography and histological analyses was performed. In addition, multiphoton autofluorescence imaging and Raman microspectroscopy analysis were applied to qualitatively and quantitatively assess the matrix integrity of the leaflets. RESULTS: Six animals from the FC group and 5 animals from the IFC group were included in the analysis. Histological explant analysis showed early inflammation in the FC valves, whereas sustainable, fully functional, devitalized acellular IFC grafts were obtained. IFC valves showed excellent haemodynamic data with fewer gradients, no pulmonary regurgitation, no calcification and acellularity. Structural remodelling of the leaflet matrix structure was only detected in FC-treated tissue, whereas IFC valves maintained matrix integrity comparable to that of native controls. The collagen crimp period and amplitude and elastin structure were significantly different in the FC valve cusps compared to IFC and native cusps. Collagen fibres in the FC valves were less aligned and straightened. CONCLUSIONS: IFC heart valves with good haemodynamic function, reduced immunogenicity and preserved matrix structures have the potential to overcome the known limitations of the clinically applied FC valve.

Surface functionalization of electrospun scaffolds using recombinant human decorin attracts circulating endothelial progenitor cells.

Decorin (DCN) is an important small leucine-rich proteoglycan present in the extracellular matrix (ECM) of many organs and tissues. Endothelial progenitor cells (EPCs) are able to interact with the surrounding ECM and bind to molecules such as DCN. Here, we recombinantly produced full-length human DCN under good laboratory practice (GLP) conditions, and after detailed immunological characterization, we investigated its potential to attract murine and human EPCs (mEPCs and hECFCs). Electrospun polymeric scaffolds were coated with DCN or stromal cell-derived factor-1 (SDF-1α) and were then dynamically cultured with both cell types. Cell viability was assessed via imaging flow cytometry. The number of captured cells was counted and compared with the non-coated controls. To characterize cell-scaffold interactions, immunofluorescence staining and scanning electron microscopy analyses were performed. We identified that DCN reduced T cell responses and attracted innate immune cells, which are responsible for ECM remodeling. A significantly higher number of EPCs attached on DCN- and SDF-1α-coated scaffolds, when compared with the uncoated controls. Interestingly, DCN showed a higher attractant effect on hECFCs than SDF-1α. Here, we successfully demonstrated DCN as promising EPC-attracting coating, which is particularily interesting when aiming to generate off-the-shelf biomaterials with the potential of in vivo cell seeding.