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Lipedema is characterized by localized accumulation of fat in the extremities, which is typically unresponsive to dietary regimens or physical activity. Although the disease is well described and has a high incidence, little is known regarding the molecular and cellular mechanisms underlying its pathogenesis. The aim of this study was to investigate the pathophysiology of lipedema adipose cells in vitro.Adipose-derived stem cells were isolated from lipoaspirates derived from lipedema and nonlipedema patients undergoing tumescent liposuction. In vitro differentiation studies were performed for up to 14 days using adipogenic or regular culture medium. Supernatants and cell lysates were tested for adiponectin, leptin, insulin-like growth factor-1, aromatase (CYP19A1), and interleukin-8 content at days 7 and 14, using enzyme-linked immunosorbent assays. Adipogenesis was evaluated by visualizing and measuring cytoplasmic lipid accumulation.Lipedema adipose-derived stem cells showed impeded adipogenesis already at early stages of in vitro differentiation. Concomitant with a strongly reduced cytoplasmic lipid accumulation, significantly lower amounts of adiponectin and leptin were detectable in supernatants from lipedema adipose-derived stem cells and adipocytes compared with control cells. In addition, lipedema and nonlipedema cells differed in their expression of insulin-like growth factor-1, aromatase (CYP19A1), and interleukin-8 and in their proliferative activity.The authors' findings indicate that in vitro adipogenesis of lipedema adipose-derived stem cells is severely hampered compared with nonlipedema adipose-derived stem cells. Lipedema adipose cells differ not only in their lipid storage capacity but also in their adipokine expression pattern. This might serve as a valuable marker for diagnosis of lipedema, probably from an early stage on.

INTRODUCTION: Metabolic diseases like diabetes mellitus often show prolonged healing and chronic wounds. Occlusive wound dressings are known to support wound closure by creating a moist environment which supports collagen synthesis, epithelialization and angiogenesis. We aimed to assess the effect of occlusion on diabetic wound fluid on the cellular level regarding fibroblast activity and angiogenetic response.MATERIAL AND METHODS: 22 split skin donor sites from 22 patients (11 patients with diabetes mellitus) were treated with occlusive dressings intraoperatively. On day 3, fluid and blood serum samples were harvested while changing the dressings. The influence of wound fluid on fibroblasts was assessed by measuring metabolic activity (Alamar Blue assay, Casey Counter), cell stress/death (LDH assay) and migration (in vitro wound healing assay) of fibroblasts. Angiogenesis of endothelial cells (HUVEC) was analyzed with the tube formation assay. Furthermore, a Magnetic Luminex Assay for multi-cytokines detection was performed focusing on inflammatory and pro-angiogenetic cytokines.RESULTS: The influence of wound fluid under occlusive dressings from diabetic patients showed a significantly increased angiogenic response and fibroblast migration compared to the non-diabetic patient group. Additionally, cell stress was increased in the diabetic group. Cytokine analysis showed an increase in VEGF-A in the diabetic group.CONCLUSION: Occlusive dressings may stimulate regenerative effects in diabetic wounds. Our in-vitro study shows the influence of wound fluid under occlusive dressings from diabetic patients on angiogenesis, migration and proliferation of fibroblasts, which are essential modulators of wound healing and scar modulation.

Background Hypoxia-inducible factor 1α (HIF-1α), a transcription factor responsible for tissue homeostasis and regeneration, presents reduced functionality in advanced age. In addition to absence of oxygen, sequestration of iron also stimulates HIF-1α. Therefore, we analyzed the efficacy of the iron-chelator deferiprone (DFP) at stimulating dermal fibroblasts. Objectives The main objective of this study was to quantify the DFP concentrations capable of stimulating dermal fibroblasts in vitro and to correlate the effective DFP concentrations with the ability of DFP to penetrate the epidermis, reach the dermis, and activate HIF-1α in vivo. Methods We measured cell proliferation, metabolic activity, HIF-1α expression, and lactate dehydrogenase levels of both young and aged fibroblasts after a 24-hour in vitro preconditioning with DFP. In addition, we evaluated cell survival rates and morphology with different cellular stainings. Finally, we performed a transdermal permeation study with a 1% DFP topical formulation to quantify the concentration required to reach the dermis. Results In vitro administration of iron-chelation therapy (156-312.5 µg/mL DFP ) on aged fibroblasts resulted in activation of various antiaging processes. The concentration required to reach the dermis within 24 hours was 1.5% (0.15 mg/mL), which corresponds well with the effective doses of our laboratory analyses. Conclusions The activation of HIF-1α by DFP enhances cell metabolism, proliferation, and survival of fibroblasts while reducing lactate dehydrogenase levels. Modulation of HIF-1α is linked to activation of key regeneration enzymes and proteins, and by proxy, antiaging. Therefore, the antiaging properties of DFP and its satisfactory dermal penetration make it a promising regenerative agent.

Surgical sutures represent the gold standard for wound closure, however, their main purpose is still limited to a mechanical function rather than playing a bioactive role. Since oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors (VEGF, PDGF-BB, or SDF-1α) at the wound site. Here, photosynthetic genetically modified microalgae were seeded in commercially available sutures and their distribution and proliferation capacity was evaluated. Additionally, the mechanical properties of seeded sutures were compared to unseeded controls that showed no significant differences. Oxygen production, as well as recombinant growth factor release was quantified in vitro over time, and confirmed that photosynthetic sutures are indeed a feasible approach for the local delivery of bioactive molecules. Finally, photosynthetic sutures were tested in order to evaluate their resistance to mechanical stress and freezing. Significant stability was observed in both conditions, and the feasibility of their use in the clinical practice was therefore confirmed. Our results suggest that photosynthetic gene therapy could be used to produce a new generation of bioactive sutures with improved healing capacities. STATEMENT OF SIGNIFICANCE: Disruption of the vascular network is intrinsic to trauma and surgery, and consequently, wound healing is characterized by diminished levels of blood perfusion. Among all the blood components, oxygen and pro-regenerative growth factors have been broadly described as key players for the healing process. Therefore, in this study we evaluated the feasibility of generating photosynthetic sutures that, in addition to mechanical fixation, could locally and stably release oxygen and recombinant human growth factors at the wound site. This novel concept has never been explored before for this type of material and represents the first attempt to create a new generation of bioactive sutures with improved regenerative capabilities.

Background: Dermal fibroblast is a powerful tool for the study of ex vivo DNA delivery in development of both cell therapy and tissue engineering products. Using genetic modification, fibroblasts can be diversely adapted and made suitable for clinical gene therapy. In this study, we first compared several non-viral transfection methods including nucleofection in rat and human primary dermal fibroblast. In addition, the original protocol for nucleofection of primary mammalian fibroblasts was modified in order to achieve the highest possible transfection efficiency, as determined by flow cytometry analysis of the green fluorescent protein (GFP) expression. Results: the results showed that transfection performance of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) yielded the best transfection efficiency with rat dermal fibroblasts and ITS (insulin, transferrin, and sodium selenite solution) was comparable to the standard nucleofection solution for human dermal fibroblasts. Conclusion: Our results suggest a promising application of the modified nucleofection method for GMP compatible therapeutic translational medical research. peerReviewed

The recent use of photosynthetic organisms such as Chlamydomonas reinhardtii in biomedical applications has demonstrated their potential for the treatment of acute and chronic tissue hypoxia. Moreover, transgenic microalgae have been suggested as an alternative in situ drug delivery system. In this study, we set out to identify the best available combination of strains and expression vectors to establish a robust platform for the expression of human pro-angiogenic growth factors, i.e., hVEGF-165, hPDGF-B, and hSDF-1, in biomedical settings. As a case study, combinations of two expression vectors (pOpt and pBC1) and two C. reinhardtii strains (UVM4 and UVM11) were compared with respect to hVEGF-165 transgene expression by determination of steady-state levels of transgenic transcripts and immunological detection of recombinant proteins produced and secreted by the generated strains. The results revealed the combination of the UVM11 strain with the pBC1 vector to be the most efficient one for high-level hVEGF-165 production. To assess the robustness of this finding, the selected combination was used to create hPDGF-B and hSDF-1 transgenic strains for optimized recombinant protein expression. Furthermore, biological activity and functionality of algal-produced recombinant pro-angiogenic growth factors were assessed by receptor phosphorylation and in vitro angiogenesis assays. The results obtained revealed a potentiating effect in the combinatorial application of transgenic strains expressing either of the three growth factors on endothelial cell tube formation ability, and thus support the idea of using transgenic algae expressing pro-angiogenic growth factors in wound healing approaches.

Summary Introduction Diabetes mellitus remains a significant public health problem, consuming over $400 billion every year. While Diabetes itself can be controlled effectively, impaired wound healing still occurs frequently in diabetic patients. Adipose-derived mesenchymal stem cells (ASCs) provide an especially appealing source for diabetic wound cell therapy. With autologous approaches, the functionality of ASCs largely underlie patient-dependent factors. Diabetes is a significant diminishing factor of MSC functionality. Here, we explore a novel strategy to enhance diabetic ASC functionality through deferoxamine (DFO) preconditioning. Material and Methods Human diabetic ASCs have been preconditioned with 150 µM and 300 µM DFO in vitro and analyzed for regenerative cytokine expression. Murine diabetic ASCs have been preconditioned with 150 µM DFO examined for their in vitro and in vivo vasculogenic capacity in Matrigel assays. Additionally, a diabetic murine wound healing model has been performed to assess the regenerative capacity of preconditioned cells. Results DFO preconditioning enhances the VEGF expression of human diabetic ASCs through hypoxia-inducible factor upregulation. The use of 150 µM of DFO was an optimal concentration to induce regenerative effects. The vasculogenic potential of preconditioned diabetic ASCs is significantly greater in vitro and in vivo. The enhanced regenerative functionality of DFO preconditioned ASCs was further confirmed in a model of diabetic murine wound healing. Conclusion These results demonstrate that DFO significantly induced the upregulation of hypoxia-inducible factor-1 alpha and VEGF in diabetic ASCs and showed efficacy in the treatment of diabetes-associated deficits of wound healing. The favorable status of DFO as a small molecule drug approved since decades for multiple indications makes this approach highly translatable.

Artículo científico The use of artificial tissues in regenerative medicine is limited due to hypoxia. As a strategy to overcome this drawback, we have shown that photosynthetic biomaterials can produce and provide oxygen independently of blood perfusion by generating chimeric animal-plant tissues during dermal regeneration. In this work, we demonstrate the safety and efficacy of photosynthetic biomaterials in vivo after engraftment in a fully immunocompetent mouse skin defect model. Further, we show that it is also possible to genetically engineer such photosynthetic scaffolds to deliver other key molecules in addition to oxygen. As a proof-of-concept, biomaterials were loaded with gene modified microalgae expressing the angiogenic recombinant protein VEGF. Survival of the algae, growth factor delivery and regenerative potential were evaluated in vitro and in vivo. This work proposes the use of photosynthetic gene therapy in regenerative medicine and provides scientific evidence for the use of engineered microalgae as an alternative to deliver recombinant molecules for gene therapy.

It has been shown that pre- and postconditioning of ischemically challenged tissue with erythropoietin (EPO) is able to reduce necrosis in a dose-dependent manner. The aim of this study was to determine the tissue-protective effects of different EPO dosages and administration regimes. Three groups of six C57Bl/6-mice each were analyzed: (1) pre- and postconditioning with initial high doses of EPO (starting at 2500 I.U./kg bw i.p.) followed by low doses of EPO (125 I.U./kg bw i.p.) (EPO-high-dose); (2) pre- and postconditioning with low doses of EPO (125 I.U./kg bw i.p.) (EPO-low-dose); and (3) untreated control group. Randomly perfused musculocutaneous flaps were mounted on dorsal skinfold chambers undergoing acute persistent ischemia and developing ∼50% necrosis without treatment. Intravital epifluorescence microscopy was performed at days 1, 3, 5, 7, and 10 after surgery, assessing flap necrosis, microcirculation, and angiogenesis. The hematocrit was measured at days 0, 3, 7, and 10. Only the EPO-low-dose regimen was associated with a significant reduction of necrosis when compared to untreated controls. EPO-low-dose showed a higher increase in both arteriolar diameter and velocity, thereby resulting in a significantly increased arteriolar blood flow and a hence higher functional capillary density (FCD) of the critically perfused zone. EPO-induced angiogenesis was significantly increased in EPO-low-dose at days 7 and 10. Only EPO-high-dose reached a significant hematocrit increase by day 10. Tissue pre- and postconditioning with low doses of EPO protects the critically perfused musculocutaneous tissue by maintaining capillary perfusion because of increased arteriolar blood flow mediated by nitric oxide (NO) expression.

Engineered tissues are highly limited by poor vascularization in vivo, leading to hypoxia. In order to overcome this challenge, we propose the use of photosynthetic biomaterials to provide oxygen. Since photosynthesis is the original source of oxygen for living organisms, we suggest that this could be a novel approach to provide a constant source of oxygen supply independently of blood perfusion. In this study we demonstrate that bioartificial scaffolds can be loaded with a solution containing the photosynthetic microalgae Chlamydomonas reinhardtii, showing high biocompatibility and photosynthetic activity in vitro. Furthermore, when photosynthetic biomaterials were engrafted in a mouse full skin defect, we observed that the presence of the microalgae did not trigger a native immune response in the host. Moreover, the analyses showed that the algae survived for at least 5 days in vivo, generating chimeric tissues comprised of algae and murine cells. The results of this study represent a crucial step towards the establishment of autotrophic tissue engineering approaches and suggest the use of photosynthetic cells to treat a broad spectrum of hypoxic conditions.

Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine.

Spatiotemporally-controlled delivery of hypoxia-induced angiogenic factor mixtures has been identified by this group as a promising strategy for overcoming the limited ability of chronically ischemic tissues to generate adaptive angiogenesis. We previously developed an implantable, as well as an injectable system for delivering fibroblast-produced factors in vivo. Here, we identify peripheral blood cells (PBCs) as the ideal factor-providing candidates, due to their autologous nature, ease of harvest and ample supply, and investigate wound-simulating biochemical and biophysical environmental parameters that can be controlled to optimize PBC angiogenic activity. It was found that hypoxia (3% O2) significantly affected the expression of a range of angiogenesis-related factors including VEGF, angiogenin and thrombospondin-1, relative to the normoxic baseline. While all three factors underwent down-regulation over time under hypoxia, there was significant variation in the temporal profile of their expression. VEGF expression was also found to be dependent on cell-scaffold material composition, with fibrin stimulating production the most, followed by collagen and polystyrene. Cell-scaffold matrix stiffness was an additional important factor, as shown by higher VEGF protein levels when PBCs were cultured on stiff vs. compliant collagen hydrogel scaffolds. Engineered PBC-derived factor mixtures could be harvested within cell-free gel and microsphere carriers. The angiogenic effectiveness of factor-loaded carriers could be demonstrated by the ability of their releasates to induce endothelial cell tubule formation and directional migration in in vitro Matrigel assays, and microvessel sprouting in the aortic ring assay. To aid the clinical translation of this approach, we propose a device design that integrates this system, and enables one-step harvesting and delivering of angiogenic factor protein mixtures from autologous peripheral blood. This will facilitate the controlled release of these factors both at the bed-side, as an angiogenic therapy in wounds and peripheral ischemic tissue, as well as pre-, intra- and post-operatively as angiogenic support for central ischemic tissue, grafts, flaps and tissue engineered implants.

While chronically ischaemic tissues are continuously exposed to hypoxia, the primary angiogenic stimulus, they fail to appropriately respond to it, as hypoxia-regulated angiogenic factor production gradually undergoes down-regulation, thus hindering adaptive angiogenesis. We have previously reported on two strategies for delivering on demand hypoxia-induced signalling (HIS) in vivo , namely, implanting living or non-viable hypoxic cell-matrix depots that actively produce factors or act as carriers of factors trapped within the matrix during in vitro pre-conditioning, respectively. This study aims to improve this approach through the development of a novel, injectable system for delivering cell-free matrix HIS-carriers. 3D spiral collagen constructs, comprising an inner cellular and outer acellular compartment, were cultured under hypoxia (5% O 2 ). Cell-produced angiogenic factors (e.g. VEGF, FGF, PLGF, IL-8) were trapped within the nano-porous matrix of the acellular compartment as they radially diffused through it. The acellular matrix was mechanically fragmented into micro-fractions and added into a low temperature (5 °C) thermo-responsive type I collagen solution, which underwent a collagen concentration-dependent solution-to-gel phase transition at 37 °C. Levels of VEGF and IL-8, delivered from matrix fractions into media by diffusion through collagen sol–gel, were up-regulated by day 4 of hypoxic culture, peaked at day 8, and gradually declined towards the baseline by day 20, while FGF levels were stable over this period. Factors captured within matrix fractions were bioactive after 3 months freeze storage, as shown by their ability to induce tubule formation in an in vitro angiogenesis assay. This system provides a minimally invasive, and repeatable, method for localised delivery of time-specific, cell-free HIS factor mixtures, as a tool for physiological induction of spatio-temporally controlled angiogenesis.

Increasing evidence suggests that vascular resident endothelial progenitor cells (VR-EPCs) are present in several organs, playing an important role in postnatal neovascularization. Here, we isolated and characterized VR-EPCs from cardiac tissue in vitro, evaluating their regenerative potential in vivo. VR-EPCs showed to be highly clonogenic and expressed several stem and differentiation markers. Under endothelial differentiation conditions, cells form capillary-like structures, in contrast to osteogenic or adipogenic differentiation conditions where no functional changes were observed. After seeding in scaffolds, cells were distributed homogeneously and directly attached to the scaffold. Then, cell seeded scaffolds were used to induce dermal regeneration in a nude mice full skin defect model. The presence of VR-EPCs enhanced dermal vascularization. Histological assays showed increased vessel number (p < 0.05) and cellularization (p < 0.05) in VR-EPCs group. In order to explore possible mechanisms of vascular regeneration, in vitro experiments were performed. Results showed that pro-angiogenic environments increased the migration capacity (p < 0.001) and ability to form capillary-like structures (p < 0.05) of VR-EPC. In addition, VR-EPCs secreted several pro-angiogenic molecules including VEGF and PDGF. These results indicate that a highly clonogenic population of VR-EPCs might be established in vitro, representing a new source for therapeutic vascularization in tissue engineering and regeneration.

Clinical success in tissue regeneration requires improvements in vascularization capacity of scaffolds. Several efforts have been made in this field including cellular and acellular technologies. In this work we combined the use of stem cells derived from pancreas or submandibular glands expressing green fluorescent protein (GFP(+)) with a commercially available scaffold for dermal regeneration. Cells were isolated, characterized and seeded in a scaffold for dermal regeneration. Scaffolds containing cells were used to induce dermal regeneration in a full skin defect model. After 3 weeks of in vivo regeneration, tissues were harvested and vascularization was analyzed. Results showed that gland-derived stem cells displayed stem cell features and presented multipotential differentiation capacity because they were able to differentiate in cell types representing the 3 different germ layers. After seeding, cells were homogeneously distributed and formed focal adhesions with the scaffold. Metabolic assays showed that cells can be cultured for at least 3 weeks in the scaffold. In vivo, the presence of pancreatic or submandibular stem cells significantly enhanced the vascularization compared to empty scaffolds. Presence of gland-derived stem cells in the regenerating tissue was confirmed by the detection of GFP expression in the wound area. In order to explore the possible mechanisms behind the improvement in vascular regeneration, in vitro experiments were performed, showing that gland-derived stem cells could contribute by angiogenic and vasculogenic mechanisms to this process. Our results suggest that the combined use of stem cells derived from glands and scaffold for dermal regeneration could be a rational alternative to improve vascularization in scaffold-mediated dermal regeneration.

The endothelium bares a paramount therapeutic and diagnostic significance in vascular disease. The current work presents a novel strategy based on the use of superparamagnetic nanoparticles to obtain an endothelial cell lining on the luminal surface of vascular conduits, which can be detected non-invasively in a clinical Magnetic Resonance Imaging (MRI) scanner. Human umbilical vein endothelial cells (HUVECs) were prelabeled with clinically approved superparamagnetic nanoparticles. Cell viability and eNOS expression were not affected by the labelling procedure. Magnetically labelled cells were delivered onto the lumen of a PTFE tubular graft by a customised electromagnet. The endothelium was detected in a 1,5T MRI scanner. Magnetic cell delivery provides an efficient technique to seed tubular scaffolds enabling the non-invasive depiction of the cells from the substrate, thus providing a reliable tool to assess the quality of cell delivery procedures.

Mesenchymal stem cells (MSCs) have been shown to improve tissue regeneration in several preclinical and clinical trials. These cells have been used in combination with three-dimensional scaffolds as a promising approach in the field of regenerative medicine. We compare the behavior of human adipose-derived MSCs (AdMSCs) on four different biomaterials that are awaiting or have already received FDA approval to determine a suitable regenerative scaffold for delivering these cells to dermal wounds and increasing healing potential. AdMSCs were isolated, characterized, and seeded onto scaffolds based on chitosan, fibrin, bovine collagen, and decellularized porcine dermis.In vitroresults demonstrated that the scaffolds strongly influence key parameters, such as seeding efficiency, cellular distribution, attachment, survival, metabolic activity, and paracrine release. Chick chorioallantoic membrane assays revealed that the scaffold composition similarly influences the angiogenic potential of AdMSCsin vivo. The wound healing potential of scaffolds increases by means of a synergistic relationship between AdMSCs and biomaterial resulting in the release of proangiogenic and cytokine factors, which is currently lacking when a scaffold alone is utilized. Furthermore, the methods used herein can be utilized to test other scaffold materials to increase their wound healing potential with AdMSCs.

The extreme dependence on external oxygen supply observed in animals causes major clinical problems and several diseases are related to low oxygen tension in tissues. The vast majority of the animals do not produce oxygen but a few exceptions have shown that photosynthetic capacity is physiologically compatible with animal life. Such symbiotic photosynthetic relationships are restricted to a few aquatic invertebrates. In this work we aimed to explore if we could create a chimerical organism by incorporating photosynthetic eukaryotic cells into a vertebrate animal model. Here, the microalgae Chlamydomonas reinhardtii was injected into zebrafish eggs and the interaction and viability of both organisms were studied. Results show that microalgae were distributed into different tissues, forming a fish-alga chimera organism for a prolonged period of time. In addition, microscopic observation of injected algae, in vivo expression of their mRNA and re-growth of the algae ex vivo suggests that they survived to the developmental process, living for several days after injection. Moreover microalgae did not trigger a significant inflammatory response in the fish. This work provides additional evidence to support the possibility that photosynthetic vertebrates can be engineered.

The identification of appropriate cell types is necessary to establish cell-based therapies in regenerative medicine. These cell types must (1) be available in an appropriate amount, (2) be easy to obtain, (3) be sufficiently expandable in vitro, and (4) fit to or at least be able to differentiate into the required cell type. Since the umbilical cord is available without any intervention and represents a notable amount of tissue, we consider it to be a promising source for isolating cells for cell-based therapies. This study demonstrates that umbilical cord stromal cells (UCSC), the connective tissue cells of the umbilical cord, can be isolated in sufficient quantities and be well expanded. UCSC feature phenotypic plasticity and thus are functionally similar to stem cells. UCSC can be differentiated into cells with osteoblastic properties (expression of alkaline phosphatase, formation of bone nodules). It is concluded that the umbilical cord should no longer be regarded as valueless tissue and be unthinkingly discarded. Instead, it should be considered a valuable resource for the isolation of potent cells for cell-based therapies, especially for treatment of bone defects.

The Schwartzwalder process was chosen for the production of ceramic TiO 2 scaffolds and showed a fully open structure with a permeability for water of 39%. The window sizes were 445 μm (45 ppi foams) and 380 μm for the 60 ppi foams. The porosity of all foams was above 78% ( n =8). It was shown that scaffolds can be produced with defined pore sizes, shape and architecture, which is a requirement for scaffold production. The macro- and microarchitecture was reproducible. Hence a reproducible ceramic scaffold processing method has been established. The interconnectivity of the pores in the scaffold was tested with a novel method. For the tests a new device was constructed where the permeability was linked to the degree of interconnectivity. Results from the permeability measurements in the mercury intrusion meter and permeability tester show that increasing pore size increases the rate of permeability. The tortuosity, which was measured in the mercury intrusion meter, was several factors higher for 60 ppi foams compared to 45 ppi and therefore also understates the lower permeability. An initial cell culture test showed that fibroblasts adhere on the foam's surface.

Turmeric, a product of Curcuma longa, has a very long history of being used for the treatment of wounds in many Asian countries. Curcumin, the principal curcuminoid of turmeric, has recently been identified as a main mediator of turmeric’s medicinal properties. However, the inherent limitations of the compound itself, such as hydrophobicity, instability, poor absorption and rapid systemic elimination, pose big hurdles for translation to wider clinical application. We present here an approach for engineering curcumin/gelatin-blended nanofibrous mats (NMs) by electrospinning to adequately enhance the bioavailability of the hydrophobic curcumin for wound repair. Curcumin was successfully formulated as an amorphous nanosolid dispersion and favorably released from gelatin-based biomimetic NMs that could be easily applied topically to experimental wounds. We show synergistic signaling by the released curcumin during the healing process: (i) mobilization of wound site fibroblasts by activating the Wnt signaling pathway, partly mediated through Dickkopf-related protein-1, and (ii) persistent inhibition of the inflammatory response through decreased expression of monocyte chemoattractant protein-1 by fibroblasts. With a combination of these effects, the curcumin/gelatin-blended NMs enhanced the regenerative process in a rat model of acute wounds, providing a method for translating this ancient medicine for use in modern wound therapy. Loading an ancient medicine onto gelatin nanofibres enhances the production of wound-healing and anti-inflammatory agents in rat models. Turmeric, the main spice in curry powder, has been used to clean and manage injuries for thousands of years. Recent studies have identified curcumin as the responsible compound for many of its medicinal properties. Now, Xinyi Dai and Arndt Schilling from Germany's Technical University of Munich and University Medical Center Gottingen and colleagues have improved curcumin's bioavailability by electrospinning it with gelatin into a tissue-mimicking network of cross-linked nanofibres. X-ray measurements showed that this procedure turned normally crystalline curcumin into an amorphous dispersion with good solubility in cellular environments. Trials on rat skins revealed that the amorphous curcumin boosted the metabolism of beneficial fibroblast cells and helped regulate anti-inflammation proteins for significantly faster wound healing. Schematic illustration of nanoformulated curcumin accelerates wound healing. (a) Curcumin nanoformulation through electrospinning greatly enhances its solubility. (b) Topically applied Curcumin/Gelatin nanofibrous mat releases bioactive curcumin to mobilize wound site fibroblasts by enhancing both cell proliferation and migration, a process partially mediated by Dickkopf-related protein-1-regulated Wnt/β-catenin pathway signaling. In the mean time, fibroblasts decreased the expression of monocyte chemoattractant protein-1, leading to lasting inhibition of inflammatory response during the healing process. As a result, wound environment turns out to be more in favor of physiological healing, with faster re-epithelialization, granulation tissue formation, as well as collagen deposition and matuaration from proliferative fibroblasts.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one’s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

Tissue engineering has opened a new therapeutic avenue that promises a revolution in regenerative medicine. To date, however, the translation of engineered tissues into clinical settings has been highly limited and the clinical results are often disappointing. Despite decades of research, the appropriate delivery of oxygen into three-dimensional cultures still remains one of the biggest unresolved problems for in vitro tissue engineering. In this work, we propose an alternative source of oxygen delivery by introducing photosynthetic scaffolds. Here we demonstrate that the unicellular and photosynthetic microalga Chlamydomonas reinhardtii can be cultured in scaffolds for tissue repair; this microalga shows high biocompatibility and photosynthetic activity. Moreover, Chlamydomonas can be co-cultured with fibroblasts, decreasing the hypoxic response under low oxygen culture conditions. Finally, results showed that photosynthetic scaffolds are capable of producing enough oxygen to be independent of external supply in vitro. The results of this study represent the first step towards engineering photosynthetic autotrophic tissues.

Adipose tissue engineering offers a promising alternative to the current surgical techniques for the treatment of soft tissue defects. It is a challenge to find the appropriate scaffold that not only represents a suitable environment for cells but also allows fabrication of customized tissue constructs, particularly in breast surgery. We investigated two different scaffolds for their potential use in adipose tissue regeneration. Sponge-like polyurethane scaffolds were prepared by mold casting with methylal as foaming agent, whereas polycaprolactone scaffolds with highly regular stacked-fiber architecture were fabricated with fused deposition modeling. Both scaffold types were seeded with human adipose tissue-derived precursor cells, cultured and implanted in nude mice using a femoral arteriovenous flow-through vessel loop for angiogenesis. In vitro, cells attached to both scaffolds and differentiated into adipocytes. In vivo, angiogenesis and adipose tissue formation were observed throughout both constructs after 2 and 4 weeks, with angiogenesis being comparable in seeded and unseeded constructs. Fibrous tissue formation and adipogenesis were more pronounced on polyurethane foam scaffolds than on polycaprolactone prototyped scaffolds. In conclusion, both scaffold designs can be effectively used for adipose tissue engineering.

Vascularization is a key process in tissue engineering and regeneration and represents one of the most important issues in the field of regenerative medicine. Thus, several strategies to improve vascularization are currently under clinical evaluation. In the present study, stem cells derived from hu[for full text, please go to the a.m. URL], 49. Jahrestagung der Österreichischen Gesellschaft für Plastische, Ästhetische und Rekonstruktive Chirurgie (ÖGPÄRC), 42. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 16. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

The use of scaffolds in skin tissue engineering is accompanied with low regeneration rates and high risk of infection. In this study, we activated an FDA-approved collagen scaffold for dermal regeneration by incorporation of copolymer-protected gene vectors (COPROGs) to induce a temporary release of[for full text, please go to the a.m. URL], 49. Jahrestagung der Österreichischen Gesellschaft für Plastische, Ästhetische und Rekonstruktive Chirurgie (ÖGPÄRC), 42. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 16. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)

Pulsed electromagnetic fields (PEMFs) have been used extensively in bone fracture repairs and wound healing. It is accepted that the induced electric field is the dose metric. The mechanisms of interaction between weak magnetic fields and biological systems present more ambiguity than that of PEMFs since weak electric currents induced by PEMFs are believed to mediate the healing process, which are absent in magnetic fields. The present study examines the response of human umbilical vein endothelial cells to weak static magnetic fields. We investigated proliferation, viability, and the expression of functional parameters such as eNOS, NO, and also gene expression of VEGF under the influence of different doses of weak magnetic fields. Applications of weak magnetic fields in tissue engineering are also discussed. Static magnetic fields may open new venues of research in the field of vascular therapies by promoting endothelial cell growth and by enhancing the healing response of the endothelium.

Optimizing seeding efficiency, reducing delayed culture periods and mimicking native tissue architecture are crucial requirements for the development of seeding procedures in tissue engineering. In vascular applications, the tubular geometry of the grafts further hampers the efficient delivery of cells onto the scaffold. To overcome these limitations, a novel technology based upon the use of magnetic fields is presented in this study: a radial magnetic force drives the cells immediately onto the luminal surface of a tubular scaffold and immobilizes the cells on the substrate’s surface promoting cell attachment. Human smooth muscle cells (SMCs) labeled with CD44 magnetic Dynabeads® were successively seeded onto the luminal surface of a tubular shaped collagen membrane. After 5 h, one additional layer of human umbilical vein endothelial cells (HUVECs) labeled with CD31 magnetic Dynabeads® was seeded onto the luminal SMCs. The co-culture was incubated during 5 days prior to analysis. Cell viability and expression profiles were preserved during the entire seeding process. Histological examination of the constructs highlighted densely packed multilayers of SMCs covered by a monolayer of endothelial cells. SEM inspection confirmed a heterotypic multilayer assembly formed by multiple layers of elongated SMCs covered by a single layer of endothelial cells. Seeding kinetics of HUVECs and SMCs showed over 90% seeding efficiency after 20 and 40 min magnetic exposure respectively. Magnetically induced cell seeding provides a valuable tool for rapid seeding procedures of tubular scaffolds while complying with the histological architecture of tissue.

We performed in situ hybridization (ISH), using radioactively labeled deoxyoligonucleotides, to localize endothelin (ET) receptor subtype mRNAs (ETA and ETB) in a series of meningiomas in comparison to normal human meninges. Our examination also addressed the cerebellum adjacent to the meninges investigated. Tumor cells of all meningiomas examined showed strong hybridization signals for ETA receptor mRNA, whereas for ETB receptor mRNA only weak hybridization signals could be detected in endothelial cells of tumor blood vessels and in some cell clusters. mRNAs for neither ET receptor were detectable in normal leptomeninges. In human cerebellum we found ETA receptor mRNA only in large blood vessels; ETB receptor mRNA was intensely expressed in Bergmann glial cells. Neither subtype was detectable in neurons.

Although bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely recognized as promising therapeutic agents, the age-related impacts on cellular function remain largely uncharacterized. In this study, we found that BM-MSCs from young donors healed wounds in a xenograft model faster compared with their aged counterparts (p < .001). Given this significant healing advantage, we then used single-cell transcriptomic analysis to provide potential molecular insights into these observations. We found that the young cells contained a higher proportion of cells characterized by a higher expression of genes involved in tissue regeneration. In addition, we identified a unique, quiescent subpopulation that was exclusively present in young donor cells. Together, these findings may explain a novel mechanism for the enhanced healing capacity of young stem cells and may have implications for autologous cell therapy in the extremes of age. Stem Cells 2019;37:240–246