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Perinatal derivatives or PnDs refer to tissues, cells and secretomes from perinatal, or birth-associated tissues. In the past 2 decades PnDs have been highly investigated for their multimodal mechanisms of action that have been exploited in various disease settings, including in different cancers and infections. Indeed, there is growing evidence that PnDs possess anticancer and antimicrobial activities, but an urgent issue that needs to be addressed is the reproducible evaluation of efficacy, both in vitro and in vivo. Herein we present the most commonly used functional assays for the assessment of antitumor and antimicrobial properties of PnDs, and we discuss their advantages and disadvantages in assessing the functionality. This review is part of a quadrinomial series on functional assays for the validation of PnDs spanning biological functions such as immunomodulation, anticancer and antimicrobial, wound healing, and regeneration.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration.

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.

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.

For more than 100 years, the human amniotic membrane (hAM) has been used in multiple tissue regeneration applications. The hAM consists of cells with stem cell characteristics and a rich layer of extracellular matrix. Undoubtedly, the hAM with viable cells has remarkable properties such as the differentiation potential into all three germ layers, immuno-modulatory, and anti-fibrotic properties. At first sight, the hAM seems to be one structural entity. However, by integrating its anatomical location, the hAM can be divided into placental, reflected, and umbilical amniotic membrane. Recent studies show that cells of these amniotic sub-regions differ considerably in their properties such as morphology, structure, and content/release of certain bioactive factors. The aim of this review is to summarize these findings and discuss the relevance of these different properties for tissue regeneration. In summary, reflected amnion seems to be more immuno-modulatory and could have a higher reprogramming efficiency, whereas placental amnion seems to be pro-inflammatory, pro-angiogenic, with higher proliferation and differentiation capacity (e.g., chondrogenic and osteogenic), and could be more suitable for certain graft constructions. Therefore, we suggest that the respective hAM sub-region should be selected in consideration of its desired outcome. This will help to optimize and fine-tune the clinical application of the hAM.

International audience; Thanks to their biological properties, amniotic membrane (AM), and its derivatives are considered as an attractive reservoir of stem cells and biological scaffolds for bone regenerative medicine. The objective of this systematic review was to assess the benefit of using AM and amniotic membrane-derived products for bone regeneration. An electronic search of the MEDLINE-Pubmed database and the Scopus database was carried out and the selection of articles was performed following PRISMA guidelines. This systematic review included 42 articles taking into consideration the studies in which AM, amniotic-derived epithelial cells (AECs), and amniotic mesenchymal stromal cells (AMSCs) show promising results for bone regeneration in animal models. Moreover, this review also presents some commercialized products derived from AM and discusses their application modalities. Finally, AM therapeutic benefit is highlighted in the reported clinical studies. This study is the first one to systematically review the therapeutic benefits of AM and amniotic membrane-derived products for bone defect healing. The AM is a promising alternative to the commercially available membranes used for guided bone regeneration. Additionally, AECs and AMSCs associated with an appropriate scaffold may also be ideal candidates for tissue engineering strategies applied to bone healing. Here, we summarized these findings and highlighted the relevance of these different products for bone regeneration.

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.

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.

Over a century ago, clinicians started to use the human amniotic membrane for coverage of wounds and burn injuries. To date, literally thousands of different clinical applications exist for this biomaterial almost exclusively in a decellularized or denuded form. Recent reconsiderations for the use of vital human amniotic membrane for clinical applications would take advantage of the versatile cells of embryonic origin including the entirety of their cell organelles. Recently, more and more evidence was found, showing mitochondria to be involved in most fundamental cellular processes, such as differentiation and cell death. In this study, we focused on specific properties of mitochondria of vital human amniotic membrane and characterized bioenergetical parameters of 2 subregions of the human amniotic membrane, the placental and reflected amnion. We found significantly different levels of adenosine triphosphate (ATP) and extracellular reactive oxygen species, concentrations of succinate dehydrogenase, and lactate upon inhibition of ATP synthase in placental and reflected amnion. We also found significantly different rates of mitochondrial respiration in isolated human amniotic epithelial cells and human amniotic mesenchymal stromal cells, according to the subregions. Differences in metabolic activities were inversely related to mitochondrial DNA copy numbers in isolated cells of placental and reflected amnion. Based on significant differences of several key parameters of energy metabolism in 2 subregions of vital amnion, we propose that these metabolic differences of vital placental and reflected amnion could have critical impact on therapeutic applications. Inclusion of region-specific metabolic properties could optimize and fine-tune the clinical application of the human amniotic membrane and improve the outcome significantly.

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.

Progress in the understanding of the biology of perinatal tissues has contributed to the breakthrough revelation of the therapeutic effects of perinatal derivatives (PnD), namely birth-associated tissues, cells, and secreted factors. The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and in vitro characterization and clinical application of PnD., Austrian Science Fund (FWF) DOC 31-B26, Medical University Graz, Universita Cattolica del Sacro Cuore, PRIN 2017 program of Italian Ministry of Research and University (MIUR) 2017RSAFK7, Ministry of Health, Italy GR-2018-12366992, Slovenian Research Agency - Slovenia P3-0108, MRIC UL IP-0510, Plan Estatal de Investigacion Cientifica y Tecnica y de Innovacion, ISCIII Subdireccion General de Evaluacion y Fomento de la Investigacion, Ministerio de Economia y Competitividad, Spain PI16/01642, European Union (EU), European Community (EC), German Research Foundation (DFG) GE-2223/2-1

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.

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

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.

Fibrin-based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid-phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non-invasive molecular imaging systems enable real-time tracking of processes in the living animal. In this study, a non-invasive fluorescence based imaging system was applied to follow and quantify site-specific degradation of fibrin sealant. To enable non-invasive tracking of fibrin in vivo, fibrin-matrix was labelled by incorporation of a fluorophore-conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore-labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16 days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site.

Human amniotic membrane (hAM) represents a tissue that is well established as biomaterial in the clinics with potential for new applications in regenerative medicine. For tissue engineering (TE) strategies, cells are usually combined with inductive factors and a carrier substrate. We have previously recognized that hAM represents a natural, preformed sheet including highly potent stem cells. In the present approach for cartilage regeneration we have induced chondrogenesis in hAM in vitro. For this, hAM biopsies were cultured for up to 56 days under chondrogenic conditions. The induced hAM was characterized for remaining viability, glycosaminoglycan (GAG) accumulation using histochemical analysis, and a quantitative assay. Collagen I, II and X was immunohistochemically determined and cartilage-specific mRNA expression of (sex determining region Y-) box 9, cartilage oligomeric matrix protein (COMP), aggrecan (AGC1), versican (CSPG2), COL1A1, COL9A2, melanoma inhibitory activity (MIA), and cartilage-linking protein 1 (CRTL1) analyzed by quantitative real-time polymerase chain reaction. Human AM was successfully induced to accumulate GAG, as demonstrated by Alcianblue staining and a significant (p < 0.001) increase of GAG/viability under chondrogenic conditions peaking in a 29.9 ± 0.9-fold induction on day 56. Further, upon chondrogenic induction collagen II positive areas were identified within histological sections and cartilage-specific markers including COMP, AGC1, CSPG2, COL1A1, COL9A2, MIA, and CRTL1 were found upregulated at mRNA level. This is the first study, demonstrating that upon in vitro induction viable human amnion expresses cartilage-specific markers and accumulates GAGs within the biomatrix. This is a promising first step towards a potential use of living hAM for cartilage TE.

Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF(164) was fused to a sequence derived from alpha 2-plasmin inhibitor (alpha(2)-PI1-8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An alpha(2)-PI1-8-fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-alpha(2)-PI1-8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-alpha(2)-PI1-8-VEGF(164) allowed exquisitely dose-dependent angiogenesis: concentrations >= 25 mu g/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01-5.0 mu g/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-alpha(2)-PI1-8-VEGF(164) was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.

Chondrogenic differentiation of adipose-derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore, quick potency assays are required to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation capacity are time-consuming, and thus, a novel enhancer and tissue-specific promoter combination was used for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were cotransfected with the Metridia luciferase-based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three-dimensional culture conditions and, in a second approach, also under two-dimensional (2D) culture conditions. With our newly developed quick potency bioassay, we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies in a very short time and at low costs.

This study evaluated the suitability of adipose-derived stem cells (ASCs) combined with fibrin matrix of variable composition for adipose tissue-equivalent formation in vitro. Therefore, undifferentiated ASCs were embedded in fibrin clots composed of 2 IU/ml thrombin and fibrinogen of varying concentrations (6.25, 12.5 and 25 mg/ml) and kept under control or adipogenic conditions. Fibrin-cell composites were evaluated by scanning electron microscopy, live/dead staining, lactate-dehydrogenase (LDH) assay, quantitative PCR for the adipogenic markers fatty acid binding protein 4 (FABP4), peroxisome proliferative activated receptor-γ (PPARγ) and leptin, leptin ELISA and oil red O staining. Cells were found homogeneously distributed throughout the clot. Their number increased to day 7 (up to 3.62-fold median) and decreased thereafter until day 28. The proliferation was unaffected by fibrinogen concentration in the control. Adipogenic conditions generally yielded higher cell numbers, which were in addition increasing with increasing fibrinogen concentrations. FABP4, PPARγ and leptin mRNA expression was strongly upregulated by adipogenic medium, which was confirmed by the levels of leptin secretion and lipid vesicles formation demonstrated by oil red O staining. When embedded in 25 mg/ml fibrinogen clots, ASCs showed the highest expression levels of FABP4 (up to 629.0-fold), PPARγ (up to 1.6-fold) and leptin (up to 57.9-fold), corroborated by significantly elevated leptin secretion (median 33.29 ng/ml) on day 14. Constructs composed of fibrin matrix of low component concentrations-allowing homogeneous cell distribution-with ASCs should represent a suitable strategy for adipose tissue formation in vivo.

Large animals such as pigs are good models for skeletal tissue engineering, since they provide physical forces similar to those of humans. Porcine bone marrow mesenchymal stem cells (BMSCs) have shown regenerative capacity similar to those of human BMSCs and can therefore be preclinically applied in settings corresponding to autologous transplantation in patients. Aiming at a one-step procedure for cartilage regeneration with autologous BMSCs, three straightforward isolation methods for BMSCs of Göttingen minipigs were compared. For this purpose, the BMSC fraction was enriched by red blood cell (RBC) lysis, dextran sedimentation or density gradient centrifugation. Isolated BMSCs were evaluated with regard to cell yield, proliferation capacity, phenotype and ability to differentiate to the chondrogenic lineage. Highest cell yields determined at the time of subcultivation were obtained using RBC lysis. In comparison, dextran sedimentation was less efficient but superior to density gradient centrifugation, which yielded significantly lower cell numbers than RBC lysis. The evaluated isolation methods resulted in cultures with equal proliferative capacity, with constant population doubling times of 50-55 h for at least 100 days (approximating to 40 cumulative population doublings) in vitro. Chondrogenic differentiation in micromass pellet cultures was evaluated by glycosaminoglycan quantification, histological staining with Alcian blue and safranin O and immunohistochemical analysis for collagen type II. These evaluations demonstrated that all three isolation methods yielded cells capable of generating cartilaginous tissue in vitro. According to our data, RBC lysis can be used to efficiently isolate porcine BMSCs in a short time frame which would allow for intraoperative one-step procedures in preclinical cartilage regeneration studies.

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca2+ dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. Statement of Significance Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry.

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.

Over the last century many studies have been performed to assess the impact of fibrin sealant (FS) components on cells. Because of the noncovalent bonding of thrombin to fibrin during fibrin clot formation, we wanted to further evaluate the impact of fibrin bound thrombin on cell viability. Initially, we quantified the activity of thrombin in three different, commercially available FS. This information was used to prepare fibrin clots covering a range of thrombin concentrations from 4 to 820 IU mL−1, but which were identical with respect to all other constituents. Although these fibrin clots did not differ in their three-dimensional structure, clots prepared with highly concentrated thrombin (820 IU mL−1) failed to support adhesion and spreading of primary human keratinocytes (NHEK). The number of attached cells was also significantly reduced on high thrombin activity clots. We hypothesized that these observations are not only the consequence of decreased proliferation but of apoptotic mechanisms, since the expression of cleaved caspase 3 and 7 was strongly enhanced on fibrin clots with high thrombin activity. This was accompanied by an induction of expression of Trail-R2 which is a receptor known to mediate apoptosis signals. Blocking of thrombin activity by hirudin led to an improvement of cell morphology and to an increase in number of attached cells. In addition, the induction of caspase 3 and 7 was also reduced. Thus, here we report for the first time that fibrin bound thrombin does not only decrease proliferation (as already published by others), it also does induce NHEK apoptosis when present at high concentrations. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.

In this study, we investigated the influence of transforming growth factor beta 3 (TGF-beta3), bone morphogenetic protein 6 (BMP-6) and basic fibroblast growth factor (FGF-2) on chondrogenesis in adipose derived stem cells (ASC). Cells were isolated from liposuction material, expanded and subjected to chondrogenic differentiation. Micromass pellets were cultured in chondrogenic medium containing 10 ng/mL TGF-beta3 which was additionally supplemented with 10 ng/mL BMP-6, 10 ng/mL FGF-2 or a combination of both. We quantitatively evaluated the cartilage specific gene expression after 14 days of culture. The end point measurements on day 35 included glycosaminoglycan (GAG) quantification, histological staining for chondrogenic markers, and transmission electron microscopy (TEM). In comparison to cultures induced with TGF-beta3/FGF-2, the presence of TGF-beta3/BMP-6 demonstrated strong induction of collagen type II, collagen type IX and aggrecan mRNA expression. This was corroborated by quantification and histological staining for GAGs and immunohistological staining for collagen II. However, when a combination of BMP-6 and FGF-2 in addition to TGF-beta3 was added, FGF-2 counteracted BMP-6, as indicated by reduced marker gene expression and weak to absent staining for GAGs. In conclusion, this study demonstrates that BMP-6 combined with TGF-beta3 is a potent inducer of chondrogenesis in human ASC. In contrast, FGF-2 does not contribute to differentiation, but rather suppresses the chondrogenic potential of BMP-6.

Preserved amniotic membrane (AM) has been used in the field of ophthalmology and wound care due to its bacteriostatic, antiphlogistic, protease-inhibiting, re-epithelialization, wound-protecting and scar formation-reducing properties. Typically, AM is applied after banking in a glycerol-preserved or freeze-dried state. Cell viabilities in different forms of preparation vary substantially, which in consequence may also be reflected in the amount and type of growth factors released from the preserved material. Therefore, we characterized the angiogenic factor (AF) profile released from different AM preparations. For this, medium was conditioned with non-preserved, glycerol- and cryo-preserved AM for 48 h, which was screened for AFs using a protein array system. In parallel, the preparations were tested for cell viability. Non-preserved as well as cryo-preserved AM maintained viabilities at 106.5 ± 23.9% and 21.9 ± 23.3%, respectively, whereas glycerol-preserved AM was found to be non-viable. Of the 20 investigated factors, high levels of angiogenin, GRO, IL-6/8, TIMP-1/2 and MCP-1 and low levels of EGF, IFNγ, IGF-1, leptin, RANTES, TGFβ1 and thrombopoietin were identified to be secreted from non-preserved AM. Cryo-preserved AM secreted high levels of IL-8, intermediate levels of GRO and TIMP-1/2 but only low levels of angiogenin, IFNγ, IL-6 and MCP-1 and no detectable EGF, IGF-1, leptin, RANTES, TGFβ1 and thrombopoietin. After banking in glycerol, AM releases only minute amounts of TIMP-1/2. Along with viability, the AF profile of amniotic membrane largely depends on the preparation method applied for banking. This should be considered for selection of an AM product for a specific clinical application. Copyright © 2009 John Wiley & Sons, Ltd.

Adipose tissue is easily available and contains high numbers of stem cells that are capable for chondrogenic differentiation. We hypothesize that a partial substitution of chondrocytes with autologous adipose-derived stem cells (ASC) might be a possible strategy to reduce the number of chondrocytes needed in matrix-associated autologous chondrocyte transplantation. To lay the ground, in vitro coculture experiments were performed using human chondrocytes and human ASC. Chondrocytes were obtained from donors undergoing matrix-associated autologous chondrocyte transplantation. ASC were isolated from liposuction material. Chondrocytes and ASC were seeded either in fibrin (Tisseel; Baxter, Vienna, Austria) or collagen matrix (Tissue Fleece; Baxter, Unterschleissheim, Germany). RNA for quantitative reverse transcriptase (RT)-polymerase chain reaction was isolated after 2 weeks of culture in chondrogenic medium, and after 4 weeks samples were processed for histology. Related to the number of chondrocytes used, coculture with ASC led to strong increase in collagen type IX mRNA expression, which is an indicator for long-term stability of cartilage. Moderate upregulation was shown for SOX9, aggrecan, melanoma inhibitory activity, cartilage link protein 1, and cartilage oligomeric matrix protein mRNA. However, expression of collagen I and collagen II indicates the synthesis of fibrous tissue, which might be due to the use of dedifferentiated chondrocytes. Tisseel provided slightly better chondrogenic conditions than Tissue Fleece. These data support the possibility to take advantage of ASC in cartilage regeneration in conjunction with autologous chondrocytes.

Cell banking of mesenchymal stem cells (SCs) from various human tissues has significantly increased the feasibility of SC-based therapies. Sources such as adipose tissue and amnion offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scaleable production and long-term analysis of these cells. Here we report the establishment and characterization of two human amnion-derived and two human adipose-derived SC lines immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT). hTERT overexpression resulted in continuously growing SC lines that were largely unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differentiation potential for up to 87 population doublings. While all generated lines showed equal immunomodulation compared to the parental cells, one of the amnion-derived immortalized lines resulted in significantly increased immunogenicity. Although telomerase proves as important tool for immortalizing cells, our data emphasize the need for careful and standardized characterization of each individual cell population for cell banks.

Fracture healing is a complicated process involving many growth factors, cells, and physical forces. In cases, where natural healing is not able, efforts have to be undertaken to improve healing. For this purpose, tissue engineering may be an option. In order to stimulate cells to form a bone tissue several factors are needed: cells, scaffold, and growth factors. Stem cells derived from bone marrow or adipose tissues are the most useful in this regard. The differentiation of the cells can be accelerated using mechanical stimulation. The first part of this chapter describes the influence of longitudinal strain application. The second part uses a sophisticated approach with stem cells on a newly developed biomaterial (Sponceram) in a rotating bed bioreactor with the administration of bone morphogenetic protein-2. It is shown that such an approach is able to produce bone tissue constructs. This may lead to production of larger constructs that can be used in clinical applications.

Thermoreversible hydrogels for tissue engineering (TE) purposes have gained increased attention in recent years as they can be combined with cells and drugs and directly injected into the body. Following the fate of transplanted cells in situ is essential in characterizing their distribution and survival, as well as the expression of specific markers or cell-matrix interactions. Existing histological embedding methods, such as paraffin wax embedding, can mechanically damage some biomaterials during processing. In this study, we describe a broadly applicable preparation protocol that allows the handling of delicate, thermoreversible scaffolds for histological sectioning. The gelatin solution permits the embedding of samples at 37 °C, which suits the solid phase of most TE scaffolds. A thermoreversible scaffold of polycaprolactone microparticles, combined with poly(polyethylene glycol methacrylate ethyl ether) and containing human adipose-derived stem cells, was prepared for histology by an initial gelatin embedding step in addition to the standard cryosectioning and paraffin processing protocols. Sections were evaluated by hematoxylin eosin staining and immunostaining for human vimentin. The gelatin embedding retained the scaffold particles and permitted the complete transfer of the construct. After rapid cooling, the solid gelatin blocks could be cryosectioned and paraffin infiltrated. In contrast to direct cryosectioning or paraffin infiltration, the extended protocol preserved the scaffold structure as well as the relevant cell epitopes, which subsequently allowed for immunostaining of human cells within the material. The gelatin embedding method proposed is a generalizable alternative to standard preparations for histological examination of a variety of delicate samples.