A three-dimensional (3D) culture system that closely replicates the in vivo microenvironment of calcifying osteoid is essential for in vitro cultivation of bone-like material. In this regard, the 3D cellulose constructs of plants may well serve as scaffolds to promote growth and differentiation of osteoblasts in culture. Our aim in this study was to generate bone-like tissue by seeding pluripotent stem cells (hiPSCs), stimulated to differentiate as osteoblasts in culture, onto the decellularised scaffolds of various plants. We then assessed expression levels of pertinent cellular markers and degrees of calcium-specific staining to gauge technical success. Apple scaffolding bearing regular pores of 300 μm seemed to provide the best construct. The bone-like tissue thus generated was implantable in a rat calvarial defect model where if helped form calcified tissue. Depending on the regularity and sizing of scaffold pores, this approach readily facilitates production of mineralized bone.

Mesenchymal stem cells (MSCs) have multiple properties including anti-inflammatory and immunomodulatory effects in various disease models and clinical treatments. These beneficial effects, however, are sometimes inconsistent and unpredictable. For wider and proper application, scientists sought to improve MSC functions by engineering. We aimed to invent a novel method to produce synthetic biological drugs from engineered MSCs. We investigated the anti-arthritic effect of engineered MSCs in a collagen-induced arthritis (CIA) model. Biologics such as etanercept are the most successful drugs used in anti-cytokine therapy. Biologics are made of protein components, and thus can be theoretically produced from cells including MSCs. MSCs were transfected with recombinant minicircles encoding etanercept (trade name, Enbrel), which is a tumour necrosis factor α blocker currently used to treat rheumatoid arthritis. We confirmed minicircle expression in MSCs in vitro based on GFP. Etanercept production was verified from the conditioned media. We confirmed that self-reproduced etanercept was biologically active in vitro. Arthritis subsided more efficiently in CIA mice injected with mcTNFR2MSCs than in those injected with conventional MSCs or etanercept only. Although this novel strategy is in a very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics and engineering MSCs.

BackgroundSkin is an organ that plays an important role as a physical barrier and has many other complex functions. Skin mimetics may be useful for studying the pathophysiology of diseases in vitro and for repairing lesions in vivo. Cord blood mononuclear cells (CBMCs) have emerged as a potential cell source for regenerative medicine. Human induced pluripotent stem cells (iPSCs) derived from CBMCs have great potential for allogenic regenerative medicine. Further study is needed on skin differentiation using CBMC-iPSCs.MethodsHuman iPSCs were generated from CBMCs by Sendai virus. CBMC-iPSCs were differentiated to fibroblasts and keratinocytes using embryonic body formation. To generate CBMC-iPSC-derived 3D skin organoid, CBMC-iPSC-derived fibroblasts were added into the insert of a Transwell plate and CBMC-iPSC-derived keratinocytes were seeded onto the fibroblast layer. Transplantation of 3D skin organoid was performed by the tie-over dressing method.ResultsEpidermal and dermal layers were developed using keratinocytes and fibroblasts differentiated from cord blood-derived human iPSCs, respectively. A complex 3D skin organoid was generated by overlaying the epidermal layer onto the dermal layer. A humanized skin model was generated by transplanting this human skin organoid into SCID mice and effectively healed skin lesions.ConclusionsThis study reveals that a human skin organoid generated using CBMC iPSCs is a novel tool for in-vitro and in-vivo dermatologic research.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0958-2) contains supplementary material, which is available to authorized users.

Epidemiological studies show an association between rheumatoid arthritis (RA) and periodontal disease. Porphyromonas gingivalis (P.gingivalis) is a well-known pathogen in periodontitis. This study investigated the pathogenic effects of P.gingivalis on autoimmune arthritis in vivo. Collagen-induced arthritis (CIA) mice were intraperitoneally injected with W83 and 2561 strains of P.gingivalis. Infection with P.gingivalis exacerbated arthritis score in CIA mice. Synovial inflammation and bone destruction in CIA mice infected with P.gingivalis were more severe than in uninfected CIA mice. Both W83 and 2561 strains were more pro-arthritic after arthritis symptom was fully activated. Interestingly, only W83 strain was arthritogenic before autoimmune reaction initiated. Citrullination was detected in synovial tissue of CIA mice and CIA mice inoculated with P.gingivalis, but not in normal control mice. The citrullinated area was greater in P.gingivalis-infected CIA mice than in non-infected CIA mice. This study showed that P.gingivalis exacerbated disease in a mouse model of autoimmune arthritis and increased the expression of citrullinated antigens in the synovium. The arthritogenic effects of P.gingivalis were at least in part, dependent upon the bacterial strain with or without fimbriae expression, route and time of infection. P.gingivalis-mediated citrullination may explain the possible link between periodontal disease and RA.

Bone destruction and inflammation are closely linked. Cytokines play an important role in inflammatory bone destruction by upregulating the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). The direct role of cytokines that act in a non-RANKL-dependent manner has yet to be elucidated. The aim of this study was to investigate the direct osteoclastogenic properties of inflammatory cytokines at different time-points of osteoclastogenesis. Mouse bone marrow macrophages were stimulated with the macrophage colony-stimulating factor (M-CSF) and various concentrations of RANKL. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-17 and IL-23, were added to the culture system of osteoclastogenesis. Two time-points of cytokine treatment were set. The ‘early’ effect of each cytokine was investigated at the time of first RANKL treatment, whereas the ‘late’ effect was investigated 48 h after the first RANKL challenge. Osteoclast differentiation and function were assessed using an osteoclast marker [tartrate-resistant acid phosphatase (TRAP)] and by visualization of pit formation. A permissive level of RANKL was required for cytokine-associated osteoclastogenesis in all experiments. In the M-CSF/RANKL monocellular culture system, IL-1β enhanced and IL-6 decreased osteoclast formation in a dose-dependent manner, regardless of temporal differences. Other cytokines showed various responses according to the phase of osteoclast maturation and the concentration of each cytokine and RANKL. Furthermore, luciferase assays showed that both IL-1β and RANKL activated the NF-κB signaling pathway. Collectively, our data revealed that targeting IL-1β may be a promising strategy to inhibit inflammation-associated bone destruction and osteoporosis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that typically results in strong inflammation and bone destruction in the joints. It is generally known that the pathogenesis of RA is linked to cardiovascular and periodontal diseases. Though rheumatoid arthritis and periodontitis share many pathologic features such as a perpetual inflammation and bone destruction, the precise mechanism underlying a link between these two diseases has not been fully elucidated. Collagen-induced arthritis (CIA) mice were orally infected with Porphyromonas gingivalis (Pg) or Pg preincubated with an anti-FimA antibody (FimA Ab) specific for fimbriae that are flexible appendages on the cell surface. Pg-infected CIA mice showed oral microbiota disruption and increased alveolar bone loss and had synovitis and joint bone destruction. However, preincubation with FimA Ab led to a significant reduction in the severity of both oral disease and arthritis. Moreover, FimA Ab attenuated bacterial attachment and aggregation on human gingival and rheumatoid arthritis synovial fibroblasts. In addition, we discovered bacteria may utilize dendritic cells, macrophages and neutrophils to migrate into the joints of CIA mice. These results suggest that disrupting Pg fimbriae function by FimA Ab ameliorates RA.

PurposeSodium chloride (NaCl) has been proposed as a driving factor in autoimmune diseases through the induction of pathogenic CD4+ T helper cells that produce interleukin-17 (Th17 cells). This study investigated the effects of NaCl on inflammatory arthritis in mice and humans.Materials and MethodsCollagen-induced arthritis (CIA) mice were fed a normal or high-salt diet ad libitum, and clinical and histologic features of arthritis were evaluated. The proportion of Th17 cells in the spleens of CIA mice fed a normal or high-salt diet was evaluated by flow cytometry, and the expression of IL-17 in joints and intestines was determined by immunohistochemical staining. We also analyzed the effect of NaCl on Th17 differentiation from peripheral blood monocytes of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and evaluated the contents of sodium and IL-17 in the synovial fluid of RA and OA patients.ResultsNaCl increased murine and human Th17 cell differentiation in a dose-dependent manner. Clinical and histological arthritis was more severe in the high-salt-fed CIA mice, compared to control CIA mice. The proportion of Th17 cells among splenocytes was higher in CIA mice fed a high-salt diet. Expression of synovial and intestinal IL-17 was also higher in high-salt-fed CIA mice. Comparison of synovial fluid between RA patients and OA patients revealed that Na+ and IL-17 were more abundant in RA synovial fluid.ConclusionThis study suggests that NaCl can aggravate arthritis by affecting Th17 differentiation. Accordingly, limiting salt intake may be helpful for treating inflammatory arthritis, such as RA.

Human bone marrow-derived mesenchymal stem cells (MSCs) have been observed to inhibit arthritis in experimental animal models such as collagen-induced arthritis. However, the exact anti-inflammatory mechanisms remain poorly understood. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine produced by immune and stromal cells. We postulated that MSCs could produce IL-1Ra and attenuate experimental arthritis. In this study, 5x106 MSCs were injected into the peritoneal cavity of IL-1Ra knockout (IL-1RaKO) mice. MSCs reduced the severity of the arthritis by histology and decreased pro-inflammatory cytokine levels in IL-1RaKO mice. The ratio of splenic T helper 17 (Th17) cells to regulatory T cells (Treg) was significantly decreased in MSC-injected IL-1RaKO mice. Purified splenic CD4+ T cells from mice in each of the treatment groups were cultured under Th17 polarizing conditions and analyzed by flow cytometry. Less expansion of the Th17 population was observed in the MSC-treated group. Interestingly, MSCs expressed inducible IL-1Ra against inflammatory environmental stimuli. Human recombinant IL-1Ra could suppress Th17 cells differentiation under Th17 polarizing conditions. These results indicate that IL-1Ra expressed by MSCs can inhibit Th17 polarization and decrease the immune response in IL-1RaKO mice. Therefore, MSC-derived IL-1Ra may inhibit inflammation in IL-1RaKO mice via effects on Th17 differentiation.