There are relatively few nanotechnologies that can produce nanocomposite scaffolds for cell growth. Electrospinning has emerged as the foremost method of producing nanofibrous biomimetic scaffolds for tissue engineering applications. In this study diamond nanoparticles were integrated into a polymer solution to develop a nanocomposite scaffold containing poly(lactide-co-glycolide) (PLGA) loaded with diamond nanoparticles. To investigate the effect of adding diamond nanoparticles to PLGA scaffolds, primary human mesenchymal stem cells (hMSCs) were seeded on the scaffolds. The cytocompatibility results showed that addition of diamond nanoparticles did not impinge upon cell proliferation, nor was there a cytotoxic cellular response after 9 days in culture. Scanning electron microscopy, transmission electron microscopy, atomic force microscopy and confocal microscopy enabled qualitative characterization of the fibres and revealed cell morphology and number. Furthermore, surface roughness was measured to evaluate diamond nanoparticle modifications, and no significant difference was found between the diamond nanocomposite and pure polymer scaffolds. On the other hand, bright spots on phase images performed by atomic force microscopy suggested a higher hardness at certain points on fibers of the PLGA-nanodiamond composites, which was supported by nanoindentation measurements. This study shows that PLGA nanofibers can be reinforced with nanodiamond without adversely affecting cell behaviour, and thus it sets the foundation for future application of these scaffolds in bone tissue engineering.

IntroductionImmature dendritic cells (DCs), specialized in the uptake of antigen, 1 reside as sentinels in almost every tissue. Under steadystate conditions, a small fraction of DCs acquire a semimature state and migrate to the draining secondary lymphoid organs. There, these semimature DCs exert tolerogenic functions by inducing apoptosis, anergy, or even a regulatory state in interacting autoreactive T cells that escaped elimination during negative thymic selection. In addition to natural regulatory T cells (Treg's), which originate in the thymus, induced Treg's, activated by tolerogenic DCs, are essential for maintainance of peripheral tolerance 2 and ensure tolerance to harmless environmental antigens. 3 In contrast, during infection, DCs engulf and process pathogenic material. Exposure to pathogen-derived molecules like lipopolysaccharide (LPS) or proinflammatory cytokines produced by cells in the microenvironment induces the full maturation of DCs, characterized by strong up-regulation of expression of costimulatory molecules and the production of proinflammatory cytokines. 4 Mature DCs constitute the most potent antigen-presenting cells, which are capable of stimulating naive antigen-specific T cells and thereby inducing a primary immune reaction.Several DC subsets with distinct phenotypes and functions have been identified. 5 In mice, CD11c has been acknowledged as a useful marker for DCs. DC subsets of myeloid origin (mDCs) mature in response to bacterial products and proinflammatory cytokines, and fully mature mDCs are inducers of strong immune responses. However, following treatment with anti-inflammatory cytokines (IL-10, transforming growth factor-␤ [TGF-␤]) or pharmacologic agents like dexamethasone (DEX) and vitamin D 3 , mDCs differentiate into a tolerogenic state. 6,7 Plasmacytoid DCs (pDCs) express markers like GR-1 and, in mice, B220, although a distinct lymphoid origin of pDCs is still a matter of debate. 8,9 pDCs have been ascribed a profound tolerogenic potential both under homeostatic conditions and even upon activation, mainly induced by DNA and RNA viruses. 10 DC functions are mainly studied using bone-marrow-derived DCs (BM-DCs) which are as potent as primary DCs isolated from spleen. 11 BM-DCs exhibit myeloid characteristics upon generation from progenitors in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas a major fraction displays a pDC-like phenotype when progenitors are cultivated with Flt3-L. 12 However, the generation of BM-DCs is time consuming, cultures may contain contaminating cell types, and BM-DCs display a heterogenous phenotype concerning the expression of major histocompatibility complex (MHC) II and costimulatory molecules, and therefore T-cell stimulatory capacity. 13 To circumvent these problems, several cell lines with DC-like characteristics have been derived either from long-term cultures of murine skin Langerhans cells 14,15 or spleen cell suspensions, 16 or by An Inside Blood analysis of this article appears at the front of this...

Background: Nanofibrous scaffolds loaded with bioactive nanoparticles are promising materials for bone tissue engineering. Methods: In this study, composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA) and diamond nanoparticles were fabricated by an electrospinning technique. PLGA was dissolved in a mixture of methylene chloride and dimethyl formamide (2:3) at a concentration of 2.3 wt%, and nanodiamond (ND) powder was added at a concentration of 0.7 wt% (about 23 wt% in dry PLGA). Results: In the composite scaffolds, the ND particles were either arranged like beads in the central part of the fibers or formed clusters protruding from the fibers. In the PLGA-ND membranes, the fibers were thicker (diameter 270 ± 9 nm) than in pure PLGA meshes (diameter 218 ± 4 nm), but the areas of pores among these fibers were smaller than in pure PLGA samples (0.46 ± 0.02 µm 2 versus 1.28 ± 0.09 µm 2 in pure PLGA samples). The PLGA-ND membranes showed higher mechanical resistance, as demonstrated by rupture tests of load and deflection of rupture probe at failure. Both types of membranes enabled the attachment, spreading, and subsequent proliferation of human osteoblast-like MG-63 cells to a similar extent, although these values were usually lower than on polystyrene dishes. Nevertheless, the cells on both types of membranes were polygonal or spindle-like in shape, and were distributed homogeneously on the samples. From days 1-7 after seeding, their number rose continuously, and at the end of the experiment, these cells were able to create a confluent layer. At the same time, the cell viability, evaluated by a LIVE/DEAD viability/cytotoxicity kit, ranged from 92% to 97% on both types of membranes. In addition, on PLGA-ND membranes, the cells formed well developed talin-containing focal adhesion plaques. As estimated by the determination of tumor necrosis factor-alpha levels in the culture medium and concentration of intercellular adhesion molecule-1, MG-63 cells, and RAW 264.7 macrophages on these membranes did not show considerable inflammatory activity. Conclusion:This study shows that nanofibrous PLGA membranes loaded with diamond nanoparticles have interesting potential for use in bone tissue engineering.

We have previously shown that particle-mediated epidermal delivery (PMED) of plasmids encoding β-galactosidase (βGal) under control of the fascin-1 promoter (pFascin-βGal) yielded selective production of the protein in skin dendritic cells (DCs), and suppressed Th2 responses in a mouse model of type I allergy by inducing Th1/Tc1 cells. However, intranasal challenge of mice immunized with pFascin-βGal induced airway hyperreactivity (AHR) and neutrophilic inflammation in the lung. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. Here we investigated the consequences of co-application of an IDO-encoding vector on the modulatory effect of DNA vaccination by PMED using pFascin-βGal in models of eosinophilic allergic and non-eosinophilic intrinsic airway inflammation. IDO-encoding plasmids and pFascin-βGal or pCMV-βGal were co-applied to abdominal skin of BALB/c mice without, before or after sensitization with βGal protein. Immune responses in the lung were analysed after intranasal provocation and airway reactivity was determined by whole body plethysmography. Co-application of pCMV-IDO with pFascin-βGal, but not pCMV-βGal inhibited the Th1/Tc1 immune response after PMED. Moreover, AHR in those mice was attenuated following intranasal challenge. Therapeutic vaccination of βGal-sensitized mice with pFascin-βGal plus pCMV-IDO slightly suppressed airway inflammation and AHR after provocation with βGal protein, while prophylactic vaccination was not effective. Altogether, our data suggest that only the combination of DC-restricted antigen and ubiquitous IDO expression attenuated asthma responses in mice, most probably by forming a tryptophan-depleted and kynurenine-enriched micromilieu known to affect neutrophils and T cells.

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