Bioactive and Elastic Emulsion Electrospun DegraPol Tubes Delivering IGF-1 for Tendon Rupture Repair.

Tendon injuries can result in two major drawbacks. Adhesions to the surrounding tissue may limit the range of motion, while fibrovascular scar formation can lead to poor biomechanical outcomes. Prosthetic devices may help to mitigate those problems. Emulsion electrospinning was used to develop a novel three-layer tube based on the polymer DegraPol (DP), with incorporated insulin-like growth factor-1 (IGF-1) in the middle layer. Scanning electron microscopy was utilized to assess the fiber diameter in IGF-1 containing pure DP meshes. Further characterization was performed with Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, and water contact angle, as well as through the assessment of mechanical properties and release kinetics from ELISA, and the bioactivity of IGF-1 by qPCR of collagen I, ki67, and tenomodulin in rabbit Achilles tenocytes. The IGF-1-containing tubes exhibited a sustained release of the growth factor up to 4 days and showed bioactivity by significantly upregulated ki67 and tenomodulin gene expression. Moreover, they proved to be mechanically superior to pure DP tubes (significantly higher fracture strain, failure stress, and elastic modulus). The novel three-layer tubes intended to be applied over conventionally sutured tendons after a rupture may help accelerate the healing process. The release of IGF-1 stimulates proliferation and matrix synthesis of cells at the repair site. In addition, adhesion formation to surrounding tissue can be reduced due to the physical barrier.

High-Affinity Cu(I)-Chelator with Potential Anti-Tumorigenic Action-A Proof-of-Principle Experimental Study of Human H460 Tumors in the CAM Assay.

Human lung cancer ranks among the most frequently treated cancers worldwide. As copper appears critical to angiogenesis and tumor growth, selective removal of copper represents a promising strategy to restrict tumor growth. To this end, we explored the activity of the novel high-affinity membrane-permeant Cu(I) chelator PSP-2 featuring a low-zeptomolar dissociation constant. Using H460 human lung cancer cells, we generated small tumors on the chorioallantoic membrane of the chicken embryo (CAM assay) and studied the effects of topical PSP-2 application on their weight and vessel density after one week. We observed a significant angiosuppression along with a marked decrease in tumor weight under PSP-2 application compared to controls. Moreover, PSP-2 exposure resulted in lower ki67+ cell numbers at a low dose but increased cell count under a high dose. Moreover, HIF-1α+ cells were significantly reduced with low-dose PSP-2 exposure compared to high-dose and control. The total copper content was considerably lower in PSP-2 treated tumors, although statistically not significant. Altogether, PSP-2 shows promising potential as an anti-cancer drug. Nevertheless, further animal experiments and application to different tumor types are mandatory to support these initial findings, paving the way toward clinical trials.

Impact of High-Molecular-Weight Hyaluronic Acid on Gene Expression in Rabbit Achilles Tenocytes In Vitro.

(1) Background: Surgical tendon repair often leads to adhesion formation, leading to joint stiffness and a reduced range of motion. Tubular implants set around sutured tendons might help to reduce peritendinous adhesions. The lubricant hyaluronic acid (HA) is a viable option for optimizing such tubes with the goal of further enhancing the anti-adhesive effect. As the implant degrades over time and diffusion is presumed, the impact of HA on tendon cells is important to know. (2) Methods: A culture medium of rabbit Achilles tenocytes was supplemented with high-molecular-weight (HMW) HA and the growth curves of the cells were assessed. Additionally, after 3, 7 and 14 days, the gene expression of several markers was analyzed for matrix assembly, tendon differentiation, fibrosis, proliferation, matrix remodeling, pro-inflammation and resolution. (3) Results: The addition of HA decreased matrix marker genes, downregulated the fibrosis marker α-SMA for a short time and slightly increased the matrix-remodeling gene MMP-2. Of the pro-inflammatory marker genes, only IL-6 was significantly upregulated. IL-6 has to be kept in check, although IL-6 is also needed for a proper initial inflammation and efficient resolution. (4) Conclusions: The observed effects in vitro support the intended anti-adhesion effect and therefore, the use of HMW HA is promising as a biodegradable implant for tendon repair.

Probing Vasoreactivity and Hypoxic Phenotype in Different Tumor Grafts Grown on the Chorioallantoic Membrane of the Chicken Embryo In Ovo Using MRI.

Tumor grafts grown on the chorioallantoic membrane (CAM) of chicken embryos represent a transition between cell culture and mammalian in vivo models. Magnetic resonance imaging (MRI) started to harness this potential. Functional gas challenge is feasible on the CAM. Using quantitative T1 and T2* mapping, we characterized the response of MC-38 colon, A549, and H460 adeno-carcinoma cell grafts to hypercapnic (HC) and hypercapnic-hyperoxic (HCHO) gas challenges, pertaining to the grafts' vascular and oxygenation phenotypes. MR imaging revealed that larger T1 and T2* were located in the center of H460 and MC-38 tumors. Quantitative analysis showed a significant reduction in T1 and a significant increase in T2* in response to HCHO for A549 grafts, while H460 and MC-38 tumors did not respond to either gas challenge. Different tumor grafts respond differentially to HC and HCHO conditions. A549 tumor grafts, with higher vessel density and smaller tumor diameter compared with H460 and MC-38 grafts, had a significant response in T1 for HCHO and T2* increased slightly during HC and significantly under HCHO, consistent with a normoxic phenotype and functional vasoreactivity. Therefore, gas challenges enable differential characterization of tumor grafts with respect to their vascular and oxygenation status.

Cell-Based HIF1α Gene Therapy Reduces Myocardial Scar and Enhances Angiopoietic Proteome, Transcriptomic and miRNA Expression in Experimental Chronic Left Ventricular Dysfunction.

Recent preclinical investigations and clinical trials with stem cells mostly studied bone-marrow-derived mononuclear cells (BM-MNCs), which so far failed to meet clinically significant functional study endpoints. BM-MNCs containing small proportions of stem cells provide little regenerative potential, while mesenchymal stem cells (MSCs) promise effective therapy via paracrine impact. Genetic engineering for rationally enhancing paracrine effects of implanted stem cells is an attractive option for further development of therapeutic cardiac repair strategies. Non-viral, efficient transfection methods promise improved clinical translation, longevity and a high level of gene delivery. Hypoxia-induced factor 1α is responsible for pro-angiogenic, anti-apoptotic and anti-remodeling mechanisms. Here we aimed to apply a cellular gene therapy model in chronic ischemic heart failure in pigs. A non-viral circular minicircle DNA vector (MiCi) was used for in vitro transfection of porcine MSCs (pMSC) with HIF1α (pMSC-MiCi-HIF-1α). pMSCs-MiCi-HIF-1α were injected endomyocardially into the border zone of an anterior myocardial infarction one month post-reperfused-infarct. Cell injection was guided via 3D-guided NOGA electro-magnetic catheter delivery system. pMSC-MiCi-HIF-1α delivery improved cardiac output and reduced myocardial scar size. Abundances of pro-angiogenic proteins were analyzed 12, 24 h and 1 month after the delivery of the regenerative substances. In a protein array, the significantly increased angiogenesis proteins were Activin A, Angiopoietin, Artemin, Endothelin-1, MCP-1; and remodeling factors ADAMTS1, FGFs, TGFb1, MMPs, and Serpins. In a qPCR analysis, increased levels of angiopeptin, CXCL12, HIF-1α and miR-132 were found 24 h after cell-based gene delivery, compared to those in untreated animals with infarction and in control animals. Expression of angiopeptin increased already 12 h after treatment, and miR-1 expression was reduced at that time point. In total, pMSC overexpressing HIF-1α showed beneficial effects for treatment of ischemic injury, mediated by stimulation of angiogenesis.

Impact of Electrospun Piezoelectric Core-Shell PVDFhfp/PDMS Mesh on Tenogenic and Inflammatory Gene Expression in Human Adipose-Derived Stem Cells: Comparison of Static Cultivation with Uniaxial Cyclic Tensile Stretching.

Specific microenvironments can trigger stem cell tenogenic differentiation, such as specific substrates or dynamic cell cultivation. Electrospun meshes composed by core-shell fibers (random or aligned; PDMS core; piezoelectric PVDFhfp shell) were fabricated by coaxial electrospinning. Elastic modulus and residual strain were assessed. Human ASCs were seeded on such scaffolds either under static conditions for 1 week or with subsequent 10% dynamic stretching for 10,800 cycles (1 Hz, 3 h), assessing load elongation curves in a Bose® bioreactor system. Gene expression for tenogenic expression, extracellular matrix, remodeling, pro-fibrotic and inflammatory marker genes were assessed (PCR). For cell-seeded meshes, the E modulus increased from 14 ± 3.8 MPa to 31 ± 17 MPa within 3 h, which was not observed for cell-free meshes. Random fibers resulted in higher tenogenic commitment than aligned fibers. Dynamic cultivation significantly enhanced pro-inflammatory markers. Compared to ASCs in culture flasks, ASCs on random meshes under static cultivation showed a significant upregulation of Mohawk, Tenascin-C and Tenomodulin. The tenogenic commitment expressed by human ASCs in contact with random PVDFhfp/PDMS paves the way for using this novel highly elastic material as an implant to be wrapped around a lacerated tendon, envisioned as a functional anti-adhesion membrane.

Electrospun tube reduces adhesion in rabbit Achilles tendon 12 weeks post-surgery without PAR-2 overexpression.

One great challenge in surgical tendon repair is the minimization of peritendinous adhesions. An electrospun tube can serve as a physical barrier around a conventionally sutured tendon. Six New Zealand White rabbits had one Achilles tendon fully transsected and sutured by a 4-strand suture. Another six rabbits had the same treatment, but with the additional electrospun DegraPol tube set around the sutured tendon. The adhesion formation to the surrounding tissue was investigated 12 weeks post-operation. Moreover, inflammation-related protease-activated receptor-2 (PAR-2) protein expression was assessed. Finally, rabbit Achilles tenocyte cultures were exposed to platelet-derived growth factor-BB (PDGF-BB), which mimicks the tendon healing environment, where PAR-2 gene expression was assessed as well as immunofluorescent staining intensity for F-actin and α-tubulin, respectively. At 12 weeks post-operation, the partially degraded DegraPol tube exhibited significantly lower adhesion formation (- 20%). PAR-2 protein expression was similar for time points 3 and 6 weeks, but increased at 12 weeks post-operation. In vitro cell culture experiments showed a significantly higher PAR-2 gene expression on day 3 after exposure to PDGF-BB, but not on day 7. The cytoskeleton of the tenocytes changed upon PDGF-BB stimulation, with signs of reorganization, and significantly decreased F-actin intensity. An electrospun DegraPol tube significantly reduces adhesion up to twelve weeks post-operation. At this time point, the tube is partially degraded, and a slight PAR-2 increase was detected in the DP treated tendons, which might however arise from particles of degrading DegraPol that were stained dark brown. PAR-2 gene expression in rabbit tenocytes reveals sensitivity at around day 10 after injury.

Suspension of Amorphous Calcium Phosphate Nanoparticles Impact Commitment of Human Adipose-Derived Stem Cells In Vitro.

Amorphous calcium phosphate (aCaP) nanoparticles may trigger the osteogenic commitment of adipose-derived stem cells (ASCs) in vitro. The ASCs of three human donors are investigated using basal culture medium DMEM to either 5 or 50 µg/mL aCaP nanoparticles suspension (control: no nanoparticles). After 7 or 14 days, stem cell marker genes, as well as endothelial, osteogenic, chondrogenic, and adipogenic genes, are analyzed by qPCR. Free calcium and phosphate ion concentrations are assessed in the cell culture supernatant. After one week and 5 µg/mL aCaP, downregulation of osteogenic markers ALP and Runx2 is found, and averaged across the three donors. Our results show that after two weeks, ALP is further downregulated, but Runx2 is upregulated. Endothelial cell marker genes, such as CD31 and CD34, are upregulated with 50 µg/mL aCaP and a 2-week exposure. Inter-donor variability is high: Two out of three donors show a significant upregulation of ALP and Runx2 at day 14 with 50 µg/mL aCaP compared to 5 µg/mL aCaP. Notably, all changes in stem cell commitment are obtained in the absence of an osteogenic medium. While the chemical composition of the culture medium and the saturation status towards calcium phosphate phases remain approximately the same for all conditions, gene expression of ASCs changes considerably. Hence, aCaP nanoparticles show the potential to trigger osteogenic and endothelial commitment in ASCs.

3D-microtissue derived secretome as a cell-free approach for enhanced mineralization of scaffolds in the chorioallantoic membrane model.

Bone regeneration is a complex process and the clinical translation of tissue engineered constructs (TECs) remains a challenge. The combination of biomaterials and mesenchymal stem cells (MSCs) may enhance the healing process through paracrine effects. Here, we investigated the influence of cell format in combination with a collagen scaffold on key factors in bone healing process, such as mineralization, cell infiltration, vascularization, and ECM production. MSCs as single cells (2D-SCs), assembled into microtissues (3D-MTs) or their corresponding secretomes were combined with a collagen scaffold and incubated on the chicken embryo chorioallantoic membrane (CAM) for 7 days. A comprehensive quantitative analysis was performed on a cellular level by histology and by microcomputed tomography (microCT). In all experimental groups, accumulation of collagen and glycosaminoglycan within the scaffold was observed over time. A pronounced cell infiltration and vascularization from the interface to the surface region of the CAM was detected. The 3D-MT secretome showed a significant mineralization of the biomaterial using microCT compared to all other conditions. Furthermore, it revealed a homogeneous distribution pattern of mineralization deposits in contrast to the cell-based scaffolds, where mineralization was only at the surface. Therefore, the secretome of MSCs assembled into 3D-MTs may represent an interesting therapeutic strategy for a next-generation bone healing concept.

MiRNA Profiles of Extracellular Vesicles Secreted by Mesenchymal Stromal Cells-Can They Predict Potential Off-Target Effects?

The cardioprotective properties of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are currently being investigated in preclinical studies. Although microRNAs (miRNAs) encapsulated in EVs have been identified as one component responsible for the cardioprotective effect of MSCs, their potential off-target effects have not been sufficiently characterized. In the present study, we aimed to investigate the miRNA profile of EVs isolated from MSCs that were derived from cord blood (CB) and adipose tissue (AT). The identified miRNAs were then compared to known targets from the literature to discover possible adverse effects prior to clinical use. Our data show that while many cardioprotective miRNAs such as miR-22-3p, miR-26a-5p, miR-29c-3p, and miR-125b-5p were present in CB- and AT-MSC-derived EVs, a large number of known oncogenic and tumor suppressor miRNAs such as miR-16-5p, miR-23a-3p, and miR-191-5p were also detected. These findings highlight the importance of quality assessment for therapeutically applied EV preparations.

Adipose tissue and the vascularization of biomaterials: Stem cells, microvascular fragments and nanofat-a review.

Tissue defects in the human body after trauma and injury require precise reconstruction to regain function. Hence, there is a great demand for clinically translatable approaches with materials that are both biocompatible and biodegradable. They should also be able to adequately integrate within the tissue through sufficient vascularization. Adipose tissue is abundant and easily accessible. It is a valuable tissue source in regenerative medicine and tissue engineering, especially with regard to its angiogenic potential. Derivatives of adipose tissue, such as microfat, nanofat, microvascular fragments, stromal vascular fraction and stem cells, are commonly used in research, but also clinically to enhance the vascularization of implants and grafts at defect sites. In plastic surgery, adipose tissue is harvested via liposuction and can be manipulated in three ways (macro-, micro- and nanofat) in the operating room, depending on its ultimate use. Whereas macro- and microfat are used as a filling material for soft tissue injuries, nanofat is an injectable viscous extract that primarily induces tissue remodeling because it is rich in growth factors and stem cells. In contrast to microfat that adds volume to a defect site, nanofat has the potential to be easily combined with scaffold materials due to its liquid and homogenous consistency and is particularly attractive for blood vessel formation. The same is true for microvascular fragments that are easily isolated from adipose tissue through collagenase digestion. In preclinical animal models, it has been convincingly shown that these vascular fragments inosculate with host vessels and subsequently accelerate scaffold perfusion and host tissue integration. Adipose tissue is also an ideal source of stem cells. It yields larger quantities of cells than any other source and is easier to access for both the patient and doctor compared with other sources such as bone marrow. They are often used for tissue regeneration in combination with biomaterials. Adipose-derived stem cells can be applied unmodified or as single cell suspensions. However, certain pretreatments, such as cultivation under hypoxic conditions or three-dimensional spheroids production, may provide substantial benefit with regard to subsequent vascularization in vivo due to induced growth factor production. In this narrative review, derivatives of adipose tissue and the vascularization of biomaterials are addressed in a comprehensive approach, including several sizes of derivatives, such as whole fat flaps for soft tissue engineering, nanofat or stem cells, their secretome and exosomes. Taken together, it can be concluded that adipose tissue and its fractions down to the molecular level promote, enhance and support vascularization of biomaterials. Therefore, there is a high potential of the individual fat component to be used in regenerative medicine.

Tumor grafts grown on the chicken chorioallantoic membrane are distinctively characterized by MRI under functional gas challenge.

Recently, a tumor model based on the chorioallantoic membrane (CAM) was characterized structurally with Magnetic Resonance Imaging (MRI). Yet, capability of MRI to assess vascular functional reserve and potential of oxygenation-sensitive MRI remain largely unexplored in this model. For this purpose, we compared MC-38 colon and A549 lung adenocarcinoma cell grafts grown on the CAM, using quantitative T1 and T2* MRI readouts as imaging markers. These are associated with vascular functionality and oxygenation status when compared between periods of air and carbogen exposure. Our data show that in A549 lung adenocarcinoma cell grafts T2* values increased significantly upon carbogen exposure (p < 0.004, Wilcoxon test; no change in T1), while MC-38 grafts displayed no changes in T1 and T2*), indicating that the grafts differ in their vascular response. Heterogeneity with regard to T1 and T2* distribution within the grafts was noted. MC-38 grafts displayed larger T1 and T2* in the graft centre, while in A549 they were distributed more towards the graft surface. Finally, qualitative assessment of gadolinium-enhancement suggests that A549 grafts display more prominent enhancement compared to MC-38 grafts. Furthermore, MC-38 grafts had 65% larger volumes than A549 grafts. Histology revealed distinct underlying phenotypes of the two tumor grafts, pertaining to the proliferative status (Ki-67) and cellularity (H&E). In sum, a functional gas challenge with carbogen is feasible through gas exchange on the CAM, and it affects MRI signals associated with vascular reactivity and oxygenation status of the tumor graft planted on the CAM. Different grafts based on A549 lung adenocarcinoma and MC-38 colon carcinoma cell lines, respectively, display distinct phenotypes that can be distinguished and characterized non-invasively in ovo using MRI in the living chicken embryo.

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration.

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell-material interaction, the material-medium interaction was also important for the stem cell commitment here, affecting the cell-medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

3D microtissue-derived human stem cells seeded on electrospun nanocomposites under shear stress: Modulation of gene expression.

OBJECTIVE: Different microenvironments trigger distinct differentiation of stem cells. Even without chemical supplementation, mechanical stimulation by shear stress may help to induce the desired differentiation. The cell format, such as three-dimensional (3D) microtissues (MTs), MT-derived cells or single cells (SCs), may have a pivotal impact as well. Here, we studied modulation of gene expression in human adipose-derived stem cells (ASCs) exposed to shear stress and/or after MT formation. MATERIALS AND METHODS: Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) at a weight ratio of 60:40 were seeded with human ASCs as MTs or as SCs and cultured in Dulbecco's modified Eagle's medium without chemical supplementation. After 2 weeks of static culture, the scaffolds were cultured statically for another 2 weeks or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm-2 min-1. Stiffness of the scaffolds was assessed as a function of time. After 4 weeks, minimum stem cell criteria markers and selected markers of osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time polymerase chain reaction. Additionally, cell distribution within the scaffolds and the allocation of the yes-associated protein (YAP) in the cells were assessed by immunohistochemistry. RESULTS: MTs decayed completely within 2 weeks after seeding on PLGA/aCaP. The osteogenic marker gene alkaline phosphatase and the endothelial cell marker gene CD31 were upregulated in MT-derived ASCs compared with SCs. Shear stress realised by fluid flow perfusion upregulated peroxisome proliferator-activated receptor gamma 2 expression in MT-derived ASCs and in SCs. The nuclear-to-cytoplasmic ratio of YAP expression was doubled under perfusion compared with that under static culture for MT-derived ASCs and SCs. CONCLUSIONS: Osteogenic and angiogenic commitments were more pronounced in MT-derived ASCs seeded on bone biomimetic electrospun nanocomposite PLGA/aCaP than in SCs seeded without induction medium. Furthermore, the static culture was superior to the perfusion regimen used here, as shear stress resulted in adipogenic commitment for MT-derived ASCs and SCs, although the YAP nuclear-to-cytoplasmic ratio indicated higher cell tensions under perfusion, usually associated with preferred osteogenic differentiation.

Novel multimodal MRI and MicroCT imaging approach to quantify angiogenesis and 3D vascular architecture of biomaterials.

Quantitative assessment of functional perfusion capacity and vessel architecture is critical when validating biomaterials for regenerative medicine purposes and requires high-tech analytical methods. Here, combining two clinically relevant imaging techniques, (magnetic resonance imaging; MRI and microcomputed tomography; MicroCT) and using the chorioallantoic membrane (CAM) assay, we present and validate a novel functional and morphological three-dimensional (3D) analysis strategy to study neovascularization in biomaterials relevant for bone regeneration. Using our new pump-assisted approach, the two scaffolds, Optimaix (laminar structure mimicking entities of the diaphysis) and DegraPol (highly porous resembling spongy bone), were shown to directly affect the architecture of the ingrowing neovasculature. Perfusion capacity (MRI) and total vessel volume (MicroCT) strongly correlated for both biomaterials, suggesting that our approach allows for a comprehensive evaluation of the vascularization pattern and efficiency of biomaterials. Being compliant with the 3R-principles (replacement, reduction and refinement), the well-established and easy-to-handle CAM model offers many advantages such as low costs, immune-incompetence and short experimental times with high-grade read-outs when compared to conventional animal models. Therefore, combined with our imaging-guided approach it represents a powerful tool to study angiogenesis in biomaterials.

High-affinity Cu(I) chelator PSP-2 as potential anti-angiogenic agent.

Copper is an essential trace metal that has been implicated in angiogenesis, the formation of new blood vessels. As tumor growth relies on establishing a functional capillary network for blood supply, copper chelation therapy may hold promise as an anti-cancer strategy by suppressing angiogenesis. To test the anti-angiogenic effect of PSP-2, a recently developed high affinity Cu(I) chelator with low zeptomolar dissociation constant, we utilized the endothelial cancer cell line EAhy926 and assessed changes in cell migration, proliferation, and tube formation in Matrigel. In addition, sprouting was assessed by the chicken and sheep aortic ring assay, and vascular pattern formation was studied in the chorioallantoic membrane of chicken embryos (CAM assay). While incubation with PSP-2 resulted in selective depletion of cellular copper levels, cell migration was not affected and the proliferating activity was even slightly increased. Moreover, the endothelial tube formation assay revealed significant morphological changes in the presence of PSP-2, with thicker tubular walls and an overall decreased meshes area. Similarly, the aortic ring assay and CAM assay showed that PSP-2 evokes significantly longer sprouts with smaller angles at branching points. Altogether, PSP-2 exhibits significant bioactivity at concentrations as low as 5 µM, rendering it a promising anti-angiogenic agent. As EAhy926 cells exhibit both endothelial and tumorigenic properties, the anti-angiogenic effect of PSP-2 might potentially translate also into anti-cancer activity.

Cellular self-assembly into 3D microtissues enhances the angiogenic activity and functional neovascularization capacity of human cardiopoietic stem cells.

While cell therapy has been proposed as next-generation therapy to treat the diseased heart, current strategies display only limited clinical efficacy. Besides the ongoing quest for the ideal cell type, in particular the very low retention rate of single-cell (SC) suspensions after delivery remains a major problem. To improve cellular retention, cellular self-assembly into 3D microtissues (MTs) prior to transplantation has emerged as an encouraging alternative. Importantly, 3D-MTs have also been reported to enhance the angiogenic activity and neovascularization potential of stem cells. Therefore, here using the chorioallantoic membrane (CAM) assay we comprehensively evaluate the impact of cell format (SCs versus 3D-MTs) on the angiogenic potential of human cardiopoietic stem cells, a promising second-generation cell type for cardiac repair. Biodegradable collagen scaffolds were seeded with human cardiopoietic stem cells, either as SCs or as 3D-MTs generated by using a modified hanging drop method. Thereafter, seeded scaffolds were placed on the CAM of living chicken embryos and analyzed for their perfusion capacity in vivo using magnetic resonance imaging assessment which was then linked to a longitudinal histomorphometric ex vivo analysis comprising blood vessel density and characteristics such as shape and size. Cellular self-assembly into 3D-MTs led to a significant increase of vessel density mainly driven by a higher number of neo-capillary formation. In contrast, SC-seeded scaffolds displayed a higher frequency of larger neo-vessels resulting in an overall 1.76-fold higher total vessel area (TVA). Importantly, despite that larger TVA in SC-seeded group, the mean perfusion capacity (MPC) was comparable between groups, therefore suggesting functional superiority together with an enhanced perfusion efficacy of the neo-vessels in 3D-MT-seeded scaffolds. This was further underlined by a 1.64-fold higher perfusion ratio when relating MPC to TVA. Our study shows that cellular self-assembly of human cardiopoietic stem cells into 3D-MTs substantially enhances their overall angiogenic potential and their functional neovascularization capacity. Hence, the concept of 3D-MTs may be considered to increase the therapeutic efficacy of future cell therapy concepts.

Modification of silicone elastomers with Bioglass 45S5® increases in ovo tissue biointegration.

Silicone is an important material family used for various medical implants. It is biocompatible, but its bioinertness prevents cell attachment, and thus tissue biointegration of silicone implants. This often results in constrictive fibrosis and implant failure. Bioglass 45S5® (BG) could be a suitable material to alter the properties of silicone, render it bioactive and improve tissue integration. Therefore, BG micro- or nanoparticles were blended into medical-grade silicone and 2D as well as 3D structures of the resulting composites were analyzed in ovo by a chick chorioallantoic membrane (CAM) assay. The biomechanical properties of the composites were measured and the bioactivity of the composites was verified in simulated body fluid. The bioactivity of BG-containing composites was confirmed visually by the formation of hydroxyapatite through scanning electron microscopy as well as by infrared spectroscopy. BG stiffens as prepared non-porous composites by 13% and 36% for micro- and nanocomposites respectively. In particular, after implantation for 7 days, the Young's modulus had increased significantly from 1.20 ± 0.01 to 1.57 ± 0.03 MPa for microcomposites and 1.44 ± 0.03 to 1.69 ± 0.29 MPa to for nanocpmosites. Still, the materials remain highly elastic and are comparably soft. The incorporation of BG into silicone overcame the bioinertness of the pure polymer. Although the overall tissue integration was weak, it was significantly improved for BG-containing porous silicones (+72% for microcomposites) and even further enhanced for composites containing nanoparticles (+94%). These findings make BG a suitable material to improve silicone implant properties. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1180-1188, 2019.