Emilia Kaivosoja

Circadian timekeeping is disturbed in rheumatoid arthritis at molecular level.

Introduction Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA. Methods Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-α. Results Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1β after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions. Conclusion We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.

Chemical and physical properties of regenerative medicine materials controlling stem cell fate

Abstract Regenerative medicine is a multidisciplinary field utilizing the potential of stem cells and the regenerative capability of the body to restore, maintain, or enhance tissue and organ functions. Stem cells are unspecialized cells that can self-renew but also differentiate into several somatic cells when subjected the appropriate environmental cues. The ability to reliably direct stem cell fate would provide tremendous potential for basic research and clinical therapies. Proper tissue function and regeneration rely on the spatial and temporal control of biophysical and biochemical cues, including soluble molecules, cell–cell contacts, cell–extracellular matrix contacts, and physical forces. The mechanisms involved remain poorly understood. This review focuses on the stem cell–extracellular matrix interactions by summarizing the observations of the effects of material variables (such as overall architecture, surface topography, charge, ζ-potential, surface energy, and elastic modulus) on the stem cell fate. It also deals with the mechanisms underlying the effects of these extrinsic, material variables. Insight in the environmental interactions of the stem cells is crucial for the development of new material-based approaches for cell culture experiments and future experimental and clinical regenerative medicine applications.

Microbial antigens mediate HLA-B27 diseases via TLRs.

HLA-B27 positive individuals are predisposed to reactive arthritis developing 1-3 weeks after urogenital and gastrointestinal infections. Also ankylosing spondylitis (AS) associates strongly to HLA-B27, but no specific infection, Klebsiella pneumoniae excluded, has been linked to it. Before the discovery of its HLA-B27 association there were many reports suggesting a link between chronic prostatitis in men or pelvic inflammatory disease in women and AS. They have since been forgotten although HLA-B27 did not help to understand, why this disease has an axial and ascending nature. It is proposed that the urogenital organs form a source of damage (or danger)-associated molecular patterns (DAMPs), either exogenous pathogen-associated molecular patterns (PAMPs) from microbes or endogenous alarmins, such as uric acid, released from necrotic cells or urate deposits. DAMPs are slowly seeded from low-down upwards via the pelvic and spinal lymphatic pathways. They reach Toll-like receptors (TLRs) in their target mesenchymal stem cells, which are stimulated to ectopic enchondral bone formation leading to syndesmophytes and bamboo spine. At the same time inflammatory cytokines induce secondary osteoporosis of the spine. This new paradigm places microbes, HLA-B27 and TLRs in the pathogenic centre stage, but without pinpointing any (one) specific pathogen; instead, shared microbial patterns are indicated.

Formation and retention of staphylococcal biofilms on DLC and its hybrids compared to metals used as biomaterials

Staphylococcus epidermidis and Staphylococcus aureus cause most of the implant-related infections. Antibiotic treatment often fails and cure requires surgical intervention. It was hypothesized that biomaterial coatings resistant to biofilms offer a preventive option. Physical vapour deposited diamond-like carbon (DLC) and its polytetrafluoroethylene (DLC-PTFE-h) and polydimethylsiloxane (DLC-PDMS-h) hybrids were compared to titanium (Ti), tantalum (Ta) and chromium (Cr) thin films on silicon wafers for their resistance against formation and/or retention of biofilms produced by S. epidermidis and S. aureus in vitro. Sample surfaces were characterized for surface topography, contact angle and zeta-potential, because such properties might affect the biofilm. Biofilm was stained using calcofluor white and analysed in fluorescence microscopy using morphometry. Sixteen hour incubation was selected in pilot tests; at this checkpoint Ti, Ta, Cr and DLC-PDMS-h were almost fully covered by biofilm, but DLC and DLC-PTFE-h were only partially biofilm coated by S. epidermidis (88±26%, p<0.001 and 56±39%, p<0.001, respectively) or S. aureus (81±24%, p<0.001 and 51±26%, p<0.001, respectively). DLC and its PTFE hybrid offer a potential biofilm hostile surface coating for implants and medical devices. This ability to resist biofilm formation and attachment could not be explained by only one factor, but it seems to be related to a combination of various properties, with electrokinetic streaming potential and protein coating being particularly important for its outcome.

Extracellular Matrix and Tissue Regeneration

Extracellular matrix (ECM) is an important component of stem cell niche areas, which provide residence, regulate stem cell pool size and control stem cell mobilization. ECM is a complex interlinked composite of collagenous molecules, non-collagenous molecules and water-rich mucopolysaccharide ground substance. Cells are integrated to their matrix via integrin and non-integrin receptors, which control adhesion, migration, division, growth, anoikis, transdifferentiation and other cellular behaviour. ECM safeguard cells and tissue architecture and strength, but also growth factor deposits, which proteinases as signalling scissors can release in a site- and process-specific manner. Selected processes, like wound healing, cartilage and heart ECM, and tumor growth are used to exemplify participation of ECM in tissue regenerative processes.

Diamond-like carbon (DLC) thin film bioelectrodes: effect of thermal post-treatments and the use of Ti adhesion layer.

The effect of thermal post-treatments and the use of Ti adhesion layer on the performance of thin film diamond like carbon bioelectrodes (DLC) have been investigated in this work. The following results were obtained: (i) The microstructure of the DLC layer after the deposition was amorphous and thermal annealing had no marked effect on the structure, (ii) formation of oxygen containing SiOx and Ti[O,C] layers were detected at the Si/Ti and Ti/DLC interfaces with the help of transmission electron microscope (TEM), (iii) thermal post-treatments increased the polar fraction of the surface energy, (iv) cyclic voltammetry (CV) measurements showed that the DLC films had wide water windows and were stable in contact with dilute sulphuric acid and phosphate buffered saline (PBS) solutions, (v) use of Ti interlayer between Pt(Ir) microwire and DLC layer was crucial for the electrodes to survive the electrochemical measurements without the loss of adhesion of the DLC layer, (vi) DLC electrodes with small exposed Pt areas were an order of magnitude more sensitive towards dopamine than Pt electrodes and (vii) thermal post-treatments did not markedly change the electrochemical behavior of the electrodes despite the significant increase in the polar nature of the surfaces. It can be concluded that thin DLC bioelectrodes are stable under physiological conditions and can detect dopamine in micro molar range, but their sensitivity must be further improved.

Osteogenesis of human mesenchymal stem cells on micro-patterned surfaces:

Osteogenic responses of human mesenchymal stromal cells (hMSCs) were compared on square-patterned, inverse square-patterned, and planar titanium, chromium, diamond-like carbon (DLC), and tantalum; hypothesis was that both the materials and patterns affect osteogenesis. Samples were produced using photolithography and physical vapor deposition. Early-marker alkaline phosphatase (ALP) and mid-markers, small body size and mothers against decapentaplegic-related protein-1 (SMAD1), runt-related transcription factor-2 (RUNX2), and osteopontin were studied using quantitative real-time polymerase chain reaction. ALP and hydroxyapatite, were colorimetrically studied. ALP reached highest values on both patterned titanium samples, but mid-markers disclosed that it was already lagging behind planar and inverse patterned tantalum. Hydroxyapatite formation disclosed that osteo-induced hMSCs passed all the differentiation stages (except on planar chromium). Presence of hydroxyapatite disclosed that both types of patterning promoted (p < 0.001) osteogenesis compared to planar samples. Results suggest that the osseocompatibility/integration of implants could be improved by changing the monotonous and featureless implant–host interface into micro-patterned interface to provide physical differentiation cues.

Cell adhesion and osteogenic differentiation on three-dimensional pillar surfaces

We hypothesized that when compared with conventional two-dimensional (2D) cultures, substrates containing 3D micropillars would allow cells to grow at levels, activating their cytoskeleton to promote osteogenesis. Fibroblasts, osteoblast-like cells, and mesenchymal stem cells (MSCs) were studied. Planar substrates were compared with 200-nm-, 5-μm-, and 20-μm-high pillars of Ormocomp®, Si, diamond-like carbon, or TiO(2). Scanning electron microscopy and staining of actin cytoskeleton showed 7.5-h adhesion to pillar edges and 5-day stretching between adhesion contacts > 100-μm distances of fibroblast and MSC in 3D networks, whereas SaOS-2 cells adhered flatly and individually on horizontal and vertical surfaces. ERK and ROCK immunostaining at 14 and 21 days confirmed activation of the cytoskeleton. In contrast to expectations, success to induce osteogenesis was dominated by the cytocompatibility of the substrate over the 3D structure. This was shown using early alkaline phosphatase, intermediate osteopontin, and late mineralization markers, together with bone nodule formation, which were seen in planar substrates and low-profile TiO(2) pillars, but were poor in the 20-μm landscape. The lack of intercellular contacts seems to halt the osteogenesis-promoting effects of cytoskeletal organization and tension described earlier.

The effect of pulsed electromagnetic fields and dehydroepiandrosterone on viability and osteo-induction of human mesenchymal stem cells.

The hypothesis of this work was that human bone marrow‐derived mesenchymal stem cells (MSCs) are regulated by pulsed electromagnetic fields (PEMFs) and by intracrine conversion of an adrenal prohormone to dihydrotestosterone. The effect of PEMF and dehydroepiandrosterone (DHEA) on viability and osteogenic differentiation of human MSCs and on the viability of osteoblastic SaOS‐2 cells was evaluated. It was found that PEMF promoted the viability rate of both cell types, whereas DHEA decreased the viability rate in a concentration‐dependent manner. PEMF did not have major effects on osteo‐induction at this low seeding density level (3000 cells/cm2). Instead, DHEA, after MSC‐mediated and 5α‐reductase‐dependent conversion to dihydrotestosterone, clearly promoted the osteo‐induction of MSCs induced with β‐glyserophosphate, ascorbate and dexamethasone. Alkaline phosphatase (ALP), SMAD1, RUNX2, osteopontin (OP) and osteocalcin (OC) RNA levels were increased and alizarin red S‐ and hydroxyapatite‐specific OsteoImageTM stainings disclosed a promoted mineralization process. In addition, DHEA increased OP and OC mRNA levels of non‐induced MSCs. A sequential use of mitogenic PEMF early during the fracture healing, followed by later administration of DHEA with osteogenic differentiating effect, might be worth subjecting to a randomized clinical trial. Copyright

IL-17C and its receptor IL-17RA/IL-17RE identify human oral epithelial cell as an inflammatory cell in recurrent aphthous ulcer

BACKGROUND Recurrent aphthous ulcer (RAU) is an ulcerative disease of non-keratinized oral mucosa. Colon and bronchial epithelial cells produce interleukin-17C (IL-17C) upon stimulation of Toll-like receptor 2 (TLR2), TLR3 and TLR5, which are highly expressed in epithelial cells in RAU lesions. We therefore investigated the eventual presence and function of IL-17C in cultured human oral keratinocytes (HOK) and control biopsies compared to RAU lesions. METHODS Expression of IL-17A, IL-17C, IL-17RA and IL-17RE was analysed in cultured HOK cells using quantitative real-time polymerase chain reaction (qRT-PCR). HOK cells were stimulated with IL-17C and analysed for IL-8 and tumour necrosis factor-α (TNF-α) using qRT-PCR. Control mucosa (n = 5) was immunostained for IL-17A, IL-17C, IL-8, TNF-α and mast cell tryptase and compared with RAU lesions (n = 5) using the mean grey scale value. RESULTS IL-17C, but no IL-17A, mRNA was found in cultured HOK cells. Components of the heterodimeric IL-17RA/IL-17RE receptor for IL-17C were also highly expressed. Stimulation of HOK with IL-17C increased TNF-α mRNA (P = 0.03; IL-8 increase was not statistically significant). HOK in RAU lesions stained intensively for IL-17C compared to controls (P = 0.006). This was associated with increased epithelial immunostaining of TNF-α (P = 0.04) and IL-8 (P = 0.02). Most of the inflammatory cells which stained for IL-17A in control mucosa and RAU lesions were also mast cell tryptase positive. CONCLUSION IL-17C is highly expressed in epithelial cells in RAU lesions, where it seems to stimulate oral keratinocytes via IL-17RA/IL-17RE to produce pro-inflammatory cytokines. Human oral epithelial cells are probably important inflammatory cells in RAU.