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Microencapsulating pancreatic islets in immunoprotective hydrogels is a promising cellular therapy for type 1 diabetes. However, a major factor limiting the encapsulated islet efficacy is inflammatory/hypoxia mediated oxidative stress, resulting in impaired insulin secretion and ultimately islet cell death. Fucoidan, a natural polysaccharide, possess strong anti-oxidant properties but its effects on beta cells and encapsulation is unknown. Here, we assessed the anti-oxidant effect of fucoidan on beta cells and its effect on encapsulated beta cell viability and function, using fucoidans extracted from two different seaweeds, namely Fucus vesiculosus (FF) and Ascophyllum nodosum (FA). FF exhibited significantly higher total antioxidant capacity and free radical scavenging activity, significantly alleviating intracellular oxidative stress in INS1E beta cells, when compared to FA. In presence of high glucose, FF fucoidans significantly increased insulin secretion both in a dose- and time-dependent manner. Viability, ATP levels and high-glucose responsiveness of rat islets encapsulated in fucoidan-containing hydrogel (Fucogel) microcapsules were significantly higher compared to those encapsulated in pure alginate microcapsules. Similar results were obtained with INS1E pseudo-islets and neonatal pig islets. Fucogels can provide a redox-modulatory niche and an immune barrier in the same time, presenting as an outstanding biomaterial for bioengineered immunoprotective beta cell replacement devices.

Summary The combination of noninvasive compositional analysis by Raman microspectrometry with high-resolution imaging in the scanning electron microscope greatly expands the analytical capabilities of the electron microscope. However, the chemical preparation of scanning electron microscope (SEM) specimens, although adequate for low-resolution imaging of superficial detail, is not the true representation of the chemistry and composition of the sample, as extraction and aggregation artefacts as a result of dehydrating and cross-linking agents are abundant. The original chemical composition and ultrastructure is only preserved using cryo preparation methods. Therefore, a complete cryo transfer flange was designed and built to add cryogenic control of specimens to the configuration of the EMRAM instrument, a combined Raman spectrometer and XL-30 ESEM instrument. The Raman spectra of two model specimen, polystyrene beads and 2.3M sucrose were studied at ambient and cryogenic temperatures as well as during a heating ramp. Comparing the fingerprint regions of polystyrene and sucrose, both measured at ambient and at cryogenic conditions, only small spectral differences were observed for the main peaks of both molecules. A pronounced sharpening of the bands occurred in the 800–400 cm−1 region, a result of the reduction of intermolecular interactions. The enhanced visibility of the lower frequency modes may offer interesting potential for more detailed interpretation of Raman spectra.

In clinical islet transplantation, allogeneic islets of Langerhans are transplanted into the portal vein of patients with type 1 diabetes, enabling the restoration of normoglycemia. After intra-hepatic transplantation several factors are involved in the decay in islet mass and function mainly caused by an immediate blood mediated inflammatory response, lack of vascularization, and allo- and autoimmunity. Bioengineered scaffolds can potentially provide an alternative extra-hepatic transplantation site for islets by improving nutrient diffusion and blood supply to the scaffold. This would ultimately result in enhanced islet viability and functionality compared to conventional intra portal transplantation. In this regard, the biomaterial choice, the three-dimensional (3D) shape and scaffold porosity are key parameters for an optimal construct design and, ultimately, transplantation outcome. We used 3D bioplotting for the fabrication of a 3D alginate-based porous scaffold as an extra-hepatic islet delivery system. In 3D-plotted alginate scaffolds the surface to volume ratio, and thus oxygen and nutrient transport, is increased compared to conventional bulk hydrogels. Several alginate mixtures have been tested for INS1E ?-cell viability. Alginate/gelatin mixtures resulted in high plotting performances, and satisfactory handling properties. INS1E ?-cells, human and mouse islets were successfully embedded in 3D-plotted constructs without affecting their morphology and viability, while preventing their aggregation. 3D plotted scaffolds could help in creating an alternative extra-hepatic transplantation site. In contrast to microcapsule embedding, in 3D plotted scaffold islets are confined in one location and blood vessels can grow into the pores of the construct, in closer contact to the embedded tissue. Once revascularization has occurred, the functionality is fully restored upon degradation of the scaffold.

In many multi-disciplinary fields of science, such as tissue engineering, where material and biological sciences are combined, there is a need for a tool that combines ultrastructural and chemical data analysis in a non-destructive manner at high resolution. We show that a combination of confocal Raman spectroscopy (CRS) and scanning electron microscopy (SEM) can be used for such analysis. Studies of atomic composition can be done by X-ray microanalysis in SEM, but this is only possible for atomic numbers greater than five and does not reveal molecular identity. Raman spectroscopy, however, can provide information on molecular composition and identity by detection of wavelength shifts caused by molecular vibrations. In this study, CRS–SEM revealed that early in vitro -formed bone extracellular matrix (ECM) produced by rat osteoprogenitor cells resembles mature bone chemically. We gained insight into the structure and chemical composition of the ECM, which was composed of mainly mineralized collagen type I fibres and areas of dense carbonated calcium phosphate related to the collagen fibre density, as revealed by Raman imaging of SEM samples. We found that CRS–SEM allows the study of specimens in a non-destructive manner and provides high-resolution structural and chemical information about inorganic and organic constituents by parallel measurements on the same sample.

Poly(ether ester amide) (PEEA) copolymers based on poly(ethylene glycol) (PEG), 1,4-butanediol and dimethyl-7,12-diaza-6,13-dione-1,18-octadecanedioate were evaluated as scaffold materials for tissue engineering. A PEEA copolymer based on PEG with a molecular weight of 300 g/mol and 25 wt% of soft segments (300 PEEA 25/75) and the parent PEA polymer (0/100) sustain the adhesion and growth of endothelial cells. The in vivo degradation of melt-pressed PEEA and PEA discs subcutaneously implanted in the back of male Wistar rats was followed up to 14 weeks. Depending on the copolymer composition, a decrease in intrinsic viscosity of about 20–30% and mass loss up to 12% were measured. During the degradation process, erosion of the surface was observed by scanning electron microscopy and light microscopy. The thermal properties of the polymers during degradation were measured by differential scanning calorimetry. During the first 2 weeks, a broadening of the melting endotherm was observed, as well as an increase in the heat of fusion. Porous matrices of PEEAs and PEA could be prepared by molding mixtures of polymer and salt particles followed by leaching of the salt.

Various diseases of the genitourinary tract can cause damage to the bladder and result in urinary retention or incontinence. Treatment options of affected bladders include augmentation cystoplasty, in which the bladder is enlarged by using a piece of intestine. Since the use of intestine in the urinary tract can cause serious adverse effects, new (bio)materials are being tested for bladder reconstruction. However, the ideal (bio)material for reconstruction of the bladder has not yet been discovered, mostly due to mechanical failure in vivo. A promising natural biomaterial is spider silk, which is outstanding in its slow degradation rate and mechanical properties. In this study, we assess the in vitro potential of spider silk as a suitable biomaterial for bladder reconstruction by cultivating primary human urothelial cells (HUCs) on native spider silk matrices. Here, we manufactured dental wire frames which were woven with the dragline silk of female Nephila Clavipes spiders under standardized conditions. Upon sterilization of spider silk frames, HUCs were cultivated on spider silk matrices and evaluated for adhesion, expansion, viability and cellular differentiation. Analysis of cell morphology and filamentous actin in HUCs revealed abundant adhesion sites and bridging of HUCs to spider silk matrices. In addition, qRT-PCRbased analysis of adhesion molecules present in HUCs revealed down-regulation of certain adhesion molecules when HUCs were cultured on spider silk. Expansion of HUCs on spider silk matrices was determined over time using Presto Blue and DAPI cell count in fields of vision and showed that spider silk supports expansion of HUCs. In vitro biocompatibility of spider silk was assessed by culturing HUCs in an extract of spider silk and revealed no cytotoxic effects on HUCs. This is supported by live/dead staining which demonstrated that HUCs were viable when cultured on spider silk matrices. QRT-PCR-based analysis of epithelial-mesenchymal transition, fibrosis and cellular differentiation markers expressed in HUCs when cultured on spider silk showed, besides down-regulation of CD44, no differences compared to control. Flow cytometry-based analysis of urothelial cellular differentiation markers CD90, CD44 and CD49f demonstrated a small subpopulation lower in CD44 expression at protein level when HUCs were cultured on spider silk. These results indicate that spider silk causes HUCs to mature slightly faster, but has no major effects on cellular differentiation of HUCs. Together, results describe a novel biomaterial which supports adhesion, expansion and viability of HUCs. Our study gives new understanding of cellular mechanisms occurring in HUCs when cultured on spider silk and demonstrates that spider silk is a promising biomaterial for bladder reconstruction.

Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.