Fabrication and Evaluation of Electrospun Silk Fibroin/Halloysite Nanotube Biomaterials for Soft Tissue Regeneration.

The production of nanofibrous materials for soft tissue repair that resemble extracellular matrices (ECMs) is challenging. Electrospinning uniquely produces scaffolds resembling the ultrastructure of natural ECMs. Herein, electrospinning was used to fabricate Bombyx mori silk fibroin (SF) and SF/halloysite nanotube (HNT) composite scaffolds. Different HNT loadings were examined, but 1 wt% HNTs enhanced scaffold hydrophilicity and water uptake capacity without loss of mechanical strength. The inclusion of 1 wt% HNTs in SF scaffolds also increased the scaffold's thermal stability without altering the molecular structure of the SF, as revealed by thermogravimetric analyses and Fourier transform infrared spectroscopy (FTIR), respectively. SF/HNT 1 wt% composite scaffolds better supported the viability and spreading of 3T3 fibroblasts and the differentiation of C2C12 myoblasts into aligned myotubes. These scaffolds coated with decellularised ECM from 3T3 cells or primary human dermal fibroblasts (HDFs) supported the growth of primary human keratinocytes. However, SF/HNT 1 wt% composite scaffolds with HDF-derived ECM provided the best microenvironment, as on these, keratinocytes formed intact monolayers with an undifferentiated, basal cell phenotype. Our data indicate the merits of SF/HNT 1 wt% composite scaffolds for applications in soft tissue repair and the expansion of primary human keratinocytes for skin regeneration.

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics.

The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell's fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

Interaction Between Skeletal Muscle Cells and Extracellular Matrix Proteins Using a Serum Free Culture System.

The ability to grow C2C12 myoblasts in a completely defined, serum free medium enables researchers to investigate the role of specific factors in myoblast proliferation, migration, fusion, and differentiation without the confounding effects of serum. The use of defined, animal free in vitro culture systems will improve reproducibility between research groups and may also enhance translation of tissue engineering techniques into clinical applications. Here, we describe the use and characterization of a serum free culture system for C2C12 myoblasts using standard tissue culture medium and readily available, defined growth factors and supplements.

Silk fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well-aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two-dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

The RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis Inactivate the Th2 cytokines interleukin-4 and interleukin-5.

The RgpA-Kgp proteinase-adhesin complexes are a primary virulence factor of Porphyromonas gingivalis, a major pathogen in the development of chronic periodontitis. The RgpA-Kgp complexes have been suggested to bias the immune response to a Th2 phenotype in disease by hydrolysis of Th1 cytokines. Here, we show that the RgpA-Kgp complexes hydrolyze and inactivate interleukin-4 (IL-4) and IL-5 under physiologically relevant conditions. Using the IL-4/IL-5-dependent TF1.8 T-cell line, it was found that at equimolar ratios of cytokine to RgpA-Kgp complexes, IL-4 and IL-5 were inactivated in the culture medium. The inactivation of IL-4 and IL-5 was RgpA-Kgp concentration dependent, as at an enzyme-to-cytokine molar ratio of 1:8, the bioactivity of the cytokines was greater than at the higher concentration of RgpA-Kgp of 1:1. Furthermore, inactivation of the cytokines by the RgpA-Kgp complexes was time dependent, as longer preincubation times resulted in lower cytokine activity. IL-5 was found to be slightly more resistant to inactivation than IL-4. Mass spectrometric analyses of IL-4 and IL-5 showed that hydrolysis by RgpA-Kgp complexes was C terminal to Arg and Lys residues of the cytokines. The peptides released indicated that the regions of IL-4 and IL-5 important for bioactivity were being hydrolyzed in the first 15 min of incubation. The ability of the RgpA-Kgp complexes to degrade Th2 cytokines may contribute to immune dysregulation and may play a role in the pathology of chronic periodontitis.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 2. Biochemical analyses.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding.

IL-2 intratumoral immunotherapy enhances CD8+ T cells that mediate destruction of tumor cells and tumor-associated vasculature: a novel mechanism for IL-2.

Therapeutic use of IL-2 can generate antitumor immunity; however, a variety of different mechanisms have been reported. We injected IL-2 intratumorally (i.t.) at different stages of growth, using our unique murine model of mesothelioma (AE17; and AE17 transfected with secretory OVA (AE17-sOVA)), and systematically analyzed real-time events as they occurred in vivo. The majority of mice with small tumors when treatment commenced displayed complete tumor regression, remained tumor free for >2 mo, and survived rechallenge with AE17 tumor cells. However, mice with large tumors at the start of treatment failed to respond. Timing experiments showed that IL-2-mediated responses were dependent upon tumor size, not on the duration of disease. Although i.t. IL-2 did not alter tumor Ag presentation in draining lymph nodes, it did enhance a previously primed, endogenous, tumor-specific in vivo CTL response that coincided with regressing tumors. Both CD4(+) and CD8(+) cells were required for IL-2-mediated tumor eradication, because IL-2 therapy failed in CD4(+)-depleted, CD8(+)-depleted, and both CD4(+)- and CD8(+)-depleted C57BL/6J animals. Tumor-infiltrating CD8(+) T cells, but not CD4(+) T cells, increased in association with a marked reduction in tumor-associated vascularity. Destruction of blood vessels required CD8(+) T cells, because this did not occur in nude mice or in CD8(+)-depleted C57BL/6J mice. These results show that repeated doses of i.t. (but not systemic) IL-2 mediates tumor regression via an enhanced endogenous tumor-specific CTL response concomitant with reduced vasculature, thereby demonstrating a novel mechanism for IL-2 activity.

Avidin is a heparin-binding protein. Affinity, specificity and structural analysis.

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.