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Developmental toxicity testing urgently requires the implementation of human relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for the human lifelong health. Fit-for-purpose NAMs for the placental-fetal interface are desirable to improve the biological knowledge of environmental exposure at molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes, as well as of the available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the developmental origins of health and disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto-placental organ-on-chip platform will be introduced as an innovative alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs (miRNAs) from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular miRNAs in concentrated conditioned medium (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEVmiRNA profiles – but not cCMmiRNAs – were impacted by lipedema. Seven sEVmiRNAs (miR–16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR–144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy individuals, whereas only one cCMmiRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

Background The interest in non-manipulated cells originating from adipose tissue has raised tremendously in the field of tissue engineering and regenerative medicine. The resulting stromal vascular fraction (SVF) cells have been successfully used in numerous clinical applications. The aim of this experimental work is, first to combine a macroporous synthetic mesh with SVF isolated using a mechanical disruption process, and to assess the effect of those cells on the early healing phase of hernia. Methods Human SVF cells combined with fibrin were used to coat commercial titanized polypropylene meshes. In vitro, viability and growth of the SVF cells were assessed using live/dead staining and scanning electron microscopy. The influence of SVF cells on abdominal wall hernia healing was conducted on immunodeficient rats, with a focus on short-term vascularization and fibrogenesis. Results Macroporous meshes were easily coated with SVF using a fibrin gel as temporary carrier. The in vitro experiments showed that the whole process including the isolation of human SVF cells and their coating on PP meshes did not impact on the SVF cells’ viability and on their capacity to attach and to proliferate. In vivo, the SVF cells were well tolerated by the animals, and coating mesh with SVF resulted in a decrease degree of vascularity compared to control group at day 21. Conclusions The utilization of SVF-coated mesh influences the level of angiogenesis during the early onset of tissue healing. Further long-term animal experiments are needed to confirm that this effect correlates with a more robust mesh integration compared to non-SVF-coated mesh.

Developmental toxicity testing urgently requires the implementation of human-relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for human lifelong health. Fit-for-purpose NAMs for the placental–fetal interface are desirable to improve the biological knowledge of environmental exposure at the molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes; in addition, we considered available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the Developmental Origins of Health and Disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto–placental organ-on-chip platform will be introduced as an innovative future alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.

Interest in mesenchymal stem cell derived extracellular vesicles (MSC‐EVs) as therapeutic agents has dramatically increased over the last decade. Current approaches to the characterization and quality control of EV‐based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label‐free immunosensing technique for characterizing clinically approved umbilical cord MSC‐EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers. Furthermore, analysis of QCM dissipation versus frequency allowed us to quantitatively determine the ratio of marker‐specific EVs versus non‐vesicular particles (NVPs) – a parameter that cannot be obtained by any other technique so far. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and NVPs in our EV preparations. This measurement modality makes it possible to identify EV sub‐fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label‐free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM‐I) and AFM measurements provides a robust multi‐marker approach to the characterization of clinically approved EV therapeutics and opens the door to improved quality control.

The prerequisite for a successful clinical use of autologous adipose-tissue-derived cells is the highest possible regenerative potential of the applied cell population, the stromal vascular fraction (SVF). Current isolation methods depend on high enzyme concentration, lysis buffer, long incubation steps and mechanical stress, resulting in single cell dissociation. The aim of the study was to limit cell manipulation and obtain a derivative comprising therapeutic cells (microtissue-SVF) without dissociation from their natural extracellular matrix, by employing a gentle good manufacturing practice (GMP)-grade isolation. The microtissue-SVF yielded larger numbers of viable cells as compared to the improved standard-SVF, both with low enzyme concentration and minimal dead cell content. It comprised stromal tissue compounds (collagen, glycosaminoglycans, fibroblasts), capillaries and vessel structures (CD31+, smooth muscle actin+). A broad range of cell types was identified by surface-marker characterisation, including mesenchymal, haematopoietic, pericytic, blood and lymphatic vascular and epithelial cells. Subpopulations such as supra-adventitial adipose-derived stromal/stem cells and endothelial progenitor cells were significantly more abundant in the microtissue-SVF, corroborated by significantly higher potency for angiogenic tube-like structure formation in vitro. The microtissue-SVF showed the characteristic phenotype and tri-lineage mesenchymal differentiation potential in vitro and an immunomodulatory and pro-angiogenic secretome. In vivo implantation of the microtissue-SVF combined with fat demonstrated successful graft integration in nude mice. The present study demonstrated a fast and gentle isolation by minor manipulation of liposuction material, achieving a therapeutically relevant cell population with high vascularisation potential and immunomodulatory properties still embedded in a fraction of its original matrix.

Background In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. Methods Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. Findings Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. Interpretation Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. Funding Austrian Research Promotion Agency FFG (“CartiScaff” #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08), Graphical abstract Image, graphical abstract

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application.

Regenerative Zellen werden zur Gewebekonstruktion und -regeneration genutzt. Dabei hat sich Fettgewebe als Quelle aufgrund guter Verfugbarkeit und leichter Gewinnung in der kosmetischen und plastischen Chirurgie durchgesetzt. Jedoch gibt es bei der Aufbereitung des zu transplantierenden Zellgemischs einiges zu beachten.

Treatment of peripheral nerve injuries has evolved over the past several decades to include the use of sophisticated new materials endowed with trophic and topographical cues that are essential for in vivo nerve fibre regeneration. In this research, we explored the use of an advanced design strategy for peripheral nerve repair, using biological and semi-synthetic hydrogels that enable controlled environmental stimuli to regenerate neurons and glial cells in a rat sciatic nerve resection model. The provisional nerve growth conduits were composed of either natural fibrin or adducts of synthetic polyethylene glycol and fibrinogen or gelatin. A photo-patterning technique was further applied to these 3D hydrogel biomaterials, in the form of laser-ablated microchannels, to provide contact guidance for unidirectional growth following sciatic nerve injury. We tested the regeneration capacity of subcritical nerve gap injuries in rats treated with photo-patterned materials and compared these with injuries treated with unpatterned hydrogels, either stiff or compliant. Among the factors tested were shear modulus, biological composition, and micropatterning of the materials. The microchannel guidance patterns, combined with appropriately matched degradation and stiffness properties of the material, proved most essential for the uniform tissue propagation during the nerve regeneration process.

Peripheral nerve fibrosis and painful adhesions are common, recurring pathological sequelae following injury. In this study, vital human amnion (hAM), an increasingly interesting biomaterial for regenerative medicine, was investigated as a novel therapy. hAM was first analyzed in vitro regarding its anti-adhesive characteristics. Then, the reflected region of hAM which was identified as more suitable, was transplanted into female Sprague Dawley rats with recurring sciatic nerve scarring (n = 24) and compared with untreated controls (n = 30) at one, four and twelve weeks. Immune response and fibrosis were investigated by (immuno)histochemical analysis. Nerve structure was examined and function determined using electrophysiology and gait analysis. Here we identified strongly reduced adhesions in the hAM-treated rats, displaying a significant difference at four weeks post transplantation compared to untreated controls (p = .0052). This correlated with the in vitro cell attachment test on hAM explants, which demonstrated a distinctly limited ability of fibroblasts to adhere to amniotic epithelial cells. Upon hAM transplantation, significantly less intraneural fibrosis was identified at the later time points. Moreover, hAM-treated rats exhibited a significantly higher sciatic functional index (SFI) after four weeks compared to controls (p .05), which indicated a potentially pro-regenerative effect of hAM. As a possible explanation, an impact of hAM on the endogenous immune response, including T cell and macrophage subsets, was indicated. We conclude that hAM is strongly effective against recurring nerve scarring and induces an anti-fibrotic and pro-regenerative effect, making it highly promising for treating adhesion-related disorders.Abnormal fibrotic bonding of tissues, frequently involving peripheral nerves, affects millions of people worldwide. These so-called adhesions usually cause severe pain and drastically reduce quality of life. To date, no adequate treatment exists and none is routinely used in the clinical practice. In this study, vital human amnion, the innermost of the fetal membranes, was transplanted in a rat model of peripheral nerve scarring and recurring adhesions as novel therapeutic approach. Amniotic cells have already demonstrated to feature stem-cell like properties and produce pro-regenerative factors, which makes the amnion an increasingly promising biomaterial for regenerative medicine. We identified that its transplantation was very effective against peripheral nerve scarring and distinctly reduced recurring adhesions. Moreover, we identified a pro-regenerative effect. This study showed that the amnion is a highly promising novel therapeutic approach for adhesion-related disorders.

One of the mainstays of facial rejuvenation strategies is volume restoration, which can be achieved by autologous fat grafting. In our novel approach, we treated the adipose tissue harvest site with extracorporeal shock wave therapy (ESWT) in order to improve the quality of the regenerative cells in situ. The latter was demonstrated by characterizing the cells of the stromal vascular fraction (SVF) in the harvested liposuction material regarding cell yield, adenosine triphosphate (ATP) content, proliferative capacity, surface marker profile, differentiation potential and secretory protein profile. Although the SVF cell yield was only slightly enhanced, viability and ATP concentration of freshly isolated cells as well as proliferation doublings after 3 weeks in culture were significantly increased in the ESWT compared with the untreated group. Likewise, cells expressing mesenchymal and endothelial/pericytic markers were significantly elevated concomitant with an improved differentiation capacity towards the adipogenic lineage and enhancement in specific angiogenic proteins. Hence, in situ ESWT might be applied in the future to promote cell fitness, adipogenesis and angiogenesis within the fat graft for successful facial rejuvenation strategies with potential long-term graft survival.

Inflammation, fibrosis and perineural adhesions with the surrounding tissue are common pathological processes following nerve injury and surgical interventions on peripheral nerves in human patients. These features can reoccur following external neurolysis, currently the most common surgical treatment for peripheral nerve scarring, thus leading to renewed nerve function impairment and chronic pain. To enable a successful evaluation of new therapeutic approaches, it is crucial to use a reproducible animal model that mimics the main clinical symptoms occurring in human patients. However, a clinically relevant model combining both histological and functional alterations has not been published to date. We therefore developed a reliable rat model that exhibits the essential pathological processes of peripheral nerve scarring. In our study, we present a novel method for the induction of nerve scarring by applying glutaraldehyde-containing glue that is known to cause nerve injury in humans. After a 3-week contact period with the sciatic nerve in female Sprague Dawley rats, we could demonstrate severe intra- and perineural scarring that resulted in grade 3 adhesions and major impairments in the electrophysiological peak amplitude compared with sham control (P=0.0478). Immunohistochemical analysis of the nerve structure revealed vigorous nerve inflammation and recruitment of T cells and macrophages. Also, distinct nerve degeneration was determined by immunostaining. These pathological alterations were further reflected in significant functional deficiencies, as determined by the analysis of relevant gait parameters as well as the quantification of the sciatic functional index starting at week 1 post-operation (P, Summary: An easily reproducible and reliable rat model for peripheral nerve scarring that allows for the effective testing of new therapeutic strategies.

Background aims Lipedema is a hormone-related disease of women characterized by enlargement of the extremities caused by subcutaneous deposition of adipose tissue. In healthy patients application of autologous adipose tissue–derived cells has shown great potential in several clinical studies for engrafting of soft tissue reconstruction in recent decades. The majority of these studies have used the stromal vascular fraction (SVF), a heterogeneous cell population containing adipose-derived stromal/stem cells (ASC), among others. Because cell identity and regenerative properties might be affected by the health condition of patients, we characterized the SVF cells of 30 lipedema patients in comparison to 22 healthy patients. Methods SVF cells were analyzed regarding cell yield, viability, adenosine triphosphate content, colony forming units and proliferative capacity, as well as surface marker profile and differentiation potential in vitro. Results Our results demonstrated a significantly enhanced SVF cell yield isolated from lipedema compared with healthy patients. In contrast, the adipogenic differentiation potential of SVF cells isolated from lipedema patients was significantly reduced compared with healthy patients. Interestingly, expression of the mesenchymal marker CD90 and the endothelial/pericytic marker CD146 was significantly enhanced when isolated from lipedema patients. Discussion The enhanced number of CD90+ and CD146+ cells could explain the increased cell yield because the other tested surface marker were not reduced in lipedema patients. Because the cellular mechanism and composition in lipedema is largely unknown, our findings might contribute to a better understanding of its etiology.

Template-mediated self-assembly synthesis has produced a diverse range of biomimetic materials with unique physicochemical properties. Here, we fabricated novel fluorescent three-dimensional (3-D) hydroxyapatite (HAP) nanorod-assembled microspheres using iron quantum cluster (FeQC) as a hybrid template, containing three organic components: hemoglobin chains, piperidine, and iron clusters. The material characterization indicated that the synthesized HAP possessed a uniform rod-like morphology, ordered 3-D architecture, high crystallinity, self-activated fluorescence, and remarkable photostability. Our study proposed that this FeQC template is a promising regulating agent to fabricate fluorescent self-assembled HAP microspheres with a controlled morphology. The effect of HAP on stem cell fate and their osteogenic differentiation was investigated by culturing human bone marrow-derived mesenchymal stromal/stem cells (BMSCs) with HAP microspheres. Significant increases in collagen matrix production and gene expression of osteogenic markers, including osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP) and alkaline phosphatase (ALP), were observed compared to the controls after 21 days of culture. Taken together, our data suggest that synthetic HAP nanorod-assembled microspheres represent a promising new biomaterial which exhibits enhanced fluorescent properties and osteoinductive effects on human BMSCs.

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular microRNAs in concentrated conditioned media (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEV miRNA profiles – but not cCM miRNAs – were impacted by lipedema. Seven sEV miRNAs (miR–16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR–144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy, whereas only one cCM miRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

Scaffold-free 3D cell cultures (e.g. pellet cultures) are widely used in medical science, including cartilage regeneration. Their drawbacks are high time/reagent consumption and lack of early readout parameters. While optimisation was achieved by automation or simplified spheroid generation, most culture systems remain expensive or require tedious procedures. The aim of this study was to establish a system for resource efficient spheroid generation with additional early readout parameters. This was achieved by a new approach for spheroid generation via self-assembly from monolayer via compartmentation of cell culture surfaces utilising laser engraving (grid plates). The compartmentation triggered contraction and rolling up of the cell monolayer, finishing in condensation into a spheroid in human adipose-derived stem cell (ASC/TERT1) and human articular chondrocytes (hACs)-ASC/TERT1 co-cultures, when cultivated on grid plates under chondrogenic conditions. Plates with 1 and 3 mm grid size yielded stable diameters (about 140 µm and 300 µm, respectively). ASC/TERT1 spheroids fully formed within 3 weeks while co-cultures took 1–2 weeks, forming significantly faster with increasing hAC ratio (p < 0.05 and 0.01 for 1:1 and 1:4 ASC/TERT1:hAC ratio, respectively). Co-cultures showed slightly lower spheroid diameters, due to earlier spheroid formation and incomplete monolayer formation. However, this was associated with a more homogeneous matrix distribution in the co-culture. Both showed differentiation capacity comparable to standard pellet culture in (immune-)histochemistry and RT-qPCR. To assess usability for cartilage repair, spheroids were embedded into a hydrogel (fibrin), yielding cellular outgrowth and matrix deposition, which was especially pronounced in co-cultures. The herein presented novel cell culture system is not only a promising tool for autonomous spheroid generation with the potential of experimental and clinical application in tissue engineering, but also for the generation of ‘building blocks’ for subsequential biofabrication strategies such as bioprinting.

Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.

Purpose Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. Results Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs’ transplanted vessels. Conclusion We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration.

Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of “AuriScaff”, an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. Statement of Significance After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches.

Background Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs. Methods Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 106) or intramyocardially (group IM, 9.88 ± 1.44 × 106). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery. In vitro bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic [fibroblast growth factor 2 (FGF2) and VEGF] were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image. Results AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min, p = 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm2, p = 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml, p = 0.006), with significant exponential decay between MMP-2 and CXCR4 (r = 0.679, p

The human amniotic membrane (hAM) has been used for tissue regeneration for over a century.In vivo(in utero), cells of the hAM are exposed to low oxygen tension (1–4% oxygen), while the hAM is usually cultured in atmospheric, meaning high, oxygen tension (20% oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5% and 20% oxygen. We found 20% oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS); however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20% oxygen. To conclude, short-time cultivation at 20% oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy.

Skeletal ischaemia-reperfusion (I-R) injury may influence patient outcome after severe vascular trauma or clamping of major vessels. The aim of this study was to observe whether locally applied vascular endothelial growth factor (VEGF) in fibrin could induce the expression of VEGF-receptor-2 (VEGFR-2) and improve the outcome after I-R injury. Transgenic mice expressing VEGFR-2 promoter-controlled luciferase were used for the assessment of VEGFR-2 expression. Ischaemia was induced for 2 h by a tension-controlled tourniquet to the hind limb, followed by 24 h of reperfusion. The animals were locally injected subcutaneously with fibrin sealant containing 20 or 200 ng VEGF; control animals received no treatment or fibrin sealant application. In vivo VEGFR-2 expression was quantified upon administration of luciferin at several observation times. For oedema and inflammation quantification, wet:dry ratio measurements and a myeloperoxidase assay of the muscle tissue were performed. Laser Doppler imaging showed that ischaemia was present and that the blood flow had returned to baseline levels after 24 h of reperfusion. VEGFR-2 expression levels in the fibrin + 200 ng VEGF were significantly higher than in all other groups. Granulocyte infiltration was reduced in both treatment groups, as well as reduced oedema formation. These results showed that VEGF released from fibrin had a positive effect on early I-R outcome in a mouse model, possibly via VEGFR-2. Copyright © 2016 John Wiley & Sons, Ltd.

Natural extracellular matrix-derived biomaterials from decellularized allogenic tissues are of increasing interest for tissue engineering because their structure and composition provide a complexity that is not achievable with current manufacturing techniques. The prerequisite to bring allogenic tissue from bench to bedside as a functional biomaterial is the full removal of cells while preserving most of its native characteristics such as structure and composition. The exceptionally dense structure of articular cartilage, however, poses a special challenge for decellularization, scaffold preparation, and reseeding. Therefore, we tested 24 different protocols aiming to remove cells and glycosaminoglycans (GAG) while preserving the collagen backbone and ultrastructure. The resulting matrices were analyzed for cell removal (DNA quantification, haematoxylin and eosin staining), GAG content (dimethyl methylene blue assay, Alcian blue staining and micro-computed tomography), collagen integrity (immunohistochemistry and ultrastructure), and biomechanics (compression test). Furthermore, seeding tests were conducted to evaluate cell viability and attachment to the scaffolds. Sodium dodecyl sulfate-based protocols yielded satisfactory reduction of DNA content, yet had negative effects on cell viability and attachment. Hydrochloric acid efficiently decellularized the scaffold and pepsin emerged as best option for GAG depletion. Combining these two reagents led to our final protocol, most efficient in DNA and GAG depletion while preserving the collagen architecture. The compressive modulus decreased in the absence of GAG to ∼1/3 of native cartilage, which is significantly higher than that by commercially available scaffolds tested as a reference (ranging from 1/25 to 1/100 of native cartilage). Cytocompatibility tests showed that human adipose-derived stromal cells readily adhered to the scaffold. In this study, we established a protocol combining freeze-thaw cycles, osmotic shock, and treatment with hydrochloric acid followed by a pepsin digestion step, achieving successful decellularization and GAG depletion within 1 week, resulting in a cytocompatible material with intact collagen structure. The protocol provides a basis for the generation of allogeneic scaffolds, potentially substituting manufactured scaffolds currently used in clinical articular cartilage treatment.

Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications.

OBJECTIVES We tested whether resuscitation supplemented with rat adipose-derived stem cells (ASCs) or secretome (conditioned media) of ASCs can ameliorate inflammation, cell/organ injury, and/or improve outcome after hemorrhagic traumatic shock (HTS). INTERVENTIONS Rats were subjected to HTS and a resuscitation protocol that mimics prehospital restrictive reperfusion followed by an adequate reperfusion phase. Twenty minutes into the restrictive reperfusion, animals received an intravenous bolus of 2 × 10 cells (ASC group) or the secretome produced by 2 × 10 ASCs/24 h (ASC-Secretome group). Controls received the vehicle (Vehicle group). All rats were observed for 28-day survival. MEASUREMENTS AND MAIN RESULTS HTS-induced inflammation represented by IL-6 was inhibited in the ASC (80%, P

Salamanders have evolved a wide variety of antipredator mechanisms and behavior patterns, including toxins and noxious or adhesive skin secretions. The high bonding strength of the natural bioadhesives makes these substances interesting for biomimetic research and applications in industrial and medical sectors. Secretions of toxic species may help to understand the direct effect of harmful substances on the cellular level. In the present study, the biocompatibility of adhesive secretions from four salamander species (Plethodon shermani, Plethodon glutinosus, Ambystoma maculatum, Ambystoma opacum) were analyzed using the MTT assay in cell culture and evaluated against toxic secretions of Pleurodeles waltl, Triturus carnifex, Pseudotriton ruber, Tylototriton verrucosus, and Salamandra salamandra. Their effect on cells was tested in direct contact (direct culture) or under the influence of the extract (indirect exposure) in accordance with the protocol of the international standard norm ISO 10993-5. Human dermal fibroblasts (NHDF), umbilical vein endothelial cells (HUVEC), and articular chondrocytes (HAC), as well as the cell lines C2C12 and L929 were used in both culture types. While the adhesive secretions from Plethodon shermani are cytocompatible and those of Ambystoma opacum are even advantageous, those of Plethodon glutinosus and Ambystoma maculatum appear to be cytotoxic to NDHF and HUVEC. Toxic secretions from Salamandra salamandra exhibited harmful effects on all cell types. Pseudotriton ruber and Triturus carnifex secretions affected certain cell types marginally; those from Pleurodeles waltl and Tylototriton verrucosus were generally well tolerated. The study shows for the first time the effect of salamander secretions on the viability of different cell types in culture. Two adhesive secretions appeared to be cell compatible and are therefore promising candidates for future investigations in the field of medical bioadhesives. Among the toxic secretions tested, only two of the five had a harmful effect on cells, indicating different cell toxicity mechanisms.