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Background: Bone repair induced by stem cells and biomaterials may represent an alternative to autologous bone grafting. Mesenchymal stromal/stem cells (MSCs), easily accessible in every human, are prototypical cells that can be tested, alone or with a biomaterial, for creating new osteoblasts. The aim of this study was to compare the efficiency of two biomaterials-biphasic calcium phosphate (BCP) and bioactive glass (BG)-when loaded with either adult bone marrow mesenchymal stem cells (BMMSCs) or newborn nasal ecto-mesenchymal stem cells (NE-MSCs), the latter being collected for further repair of lip cleft-associated bone loss. Materials and Methods: BMMSCs were collected from two adults and NE-MSCs from two newborn infants. An in vitro study was performed in order to determine the best experimental conditions for adhesion, viability, proliferation and osteoblastic differentiation on BCP or BG granules. Bone-associated morphological changes and gene expression modifications were quantified using histological and molecular techniques. The in vivo study was based on the subcutaneous implantation in nude mice of the biomaterials, loaded or not with one of the two cell types. Eight weeks after, bone formation was assessed using histological and electron microscopy techniques. Results: Both cell types-BMMSC and NE-MSC-display the typical stem cell surface markers-CD73+, CD90+, CD105+, nestin - and exhibit the MSC-associated osteogenic, chondrogenic and adipogenic multipotency. NE-MSCs produce less collagen and alkaline phosphatase than BMMSCs. At the transcript level, NE-MSCs express more abundantly three genes coding for bone sialoprotein, osteocalcin and osteopontin while BMMSCs produce extra copies of RunX2. BMMSCs and NE-MSCs adhere and survive on BCP and BG. In vivo experiments reveal that bone formation is only observed with BMMSCs transplanted on BCP biomaterial. Conclusion: Although belonging to the same superfamily of mesenchymal stem cells, BMMSCs and NE-MSCs exhibit striking differences, in vitro and in vivo . For future clinical applications, the association of BMMSCs with BCP biomaterial seems to be the most promising., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fievet, Serratrice, Brulin, Giraudo, Véran, Degardin, Sabatier, Féron and Layrolle.)

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. STATEMENT OF SIGNIFICANCE: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Improvements in the clinical outcome of osteosarcoma have plateaued in recent decades with poor translation between preclinical testing and clinical efficacy. Organotypic cultures retain key features of patient tumours, such as a myriad of cell types organized within an extracellular matrix, thereby presenting a more realistic and personalised screening of chemotherapeutic agents ex vivo. To test this concept for the first time in osteosarcoma, murine and canine osteosarcoma organotypic models were maintained for up to 21 days and in-depth analysis identified proportions of immune and stromal cells present at levels comparable to that reported in vivo in the literature. Cytotoxicity testing of a range of chemotherapeutic drugs (mafosfamide, cisplatin, methotrexate, etoposide, and doxorubicin) on murine organotypic culture ex vivo found limited response to treatment, with immune and stromal cells demonstrating enhanced survival over the global tumour cell population. Furthermore, significantly decreased sensitivity to a range of chemotherapeutics in 3D organotypic culture relative to 2D monolayer was observed, with subsequent investigation confirming reduced sensitivity in 3D than in 2D, even at equivalent levels of drug uptake. Finally, as proof of concept for the application of this model to personalised drug screening, chemotherapy testing with doxorubicin was performed on biopsies obtained from canine osteosarcoma patients. Together, this study highlights the importance of recapitulating the 3D tumour multicellular microenvironment to better predict drug response and provides evidence for the utility and possibilities of organotypic culture for enhanced preclinical selection and evaluation of chemotherapeutics targeting osteosarcoma.

In bone regeneration induced by the combination of mesenchymal stromal cells (MSCs) and calcium-phosphate (CaP) materials, osteoclasts emerge as a pivotal cell linking inflammation and bone formation. Favorable outcomes are observed despite short-term engraftments of implanted MSCs, highlighting their major paracrine function and the possible implication of cell death in modulating their secretions. In this work, we focused on the communication from MSCs towards osteoclasts-like cells in vitro. MSCs seeded on a CaP biomaterial or undergoing induced apoptosis produced a conditioned media favoring the development of osteoclasts from human CD14+ monocytes. On the contrary, MSCs' apoptotic secretion inhibited the development of inflammatory multinucleated giant cells formed after IL-4 stimulation. Components of MSCs' secretome before and after apoptotic stress were compared using mass spectrometry-based quantitative proteomics and a complementary immunoassay for major cytokines. CXCR-1 and CXCR-2 ligands, primarily IL-8/CXCL-8 but also the growth-regulated proteins CXCL-1, -2 or -3, were suggested as the major players of MSCs' pro-osteoclastic effect. These findings support the hypothesis that osteoclasts are key players in bone regeneration and suggest that apoptosis plays an important role in MSCs' effectiveness.

Progastrin is an unprocessed soluble peptide precursor with a well-described tumor-promoting role in colorectal cancer. It is expressed at small levels in the healthy intestinal mucosa, and its expression is enhanced at early stages of intestinal tumor development, with high levels of this peptide in hyperplastic intestinal polyps being associated with poor neoplasm-free survival in patients. Yet, the precise type of progastrin-producing cells in the healthy intestinal mucosa and in early adenomas remains unclear. Here, we used a combination of immunostaining, RNAscope labelling and retrospective analysis of single cell RNAseq results to demonstrate that progastrin is produced within intestinal crypts by a subset of Bmi1 + /Prox1 + /LGR5 low endocrine cells, previously shown to act as replacement stem cells in case of mucosal injury. In contrast, our findings indicate that intestinal stem cells, specified by expression of the Wnt signaling target LGR5, become the main source of progastrin production in early mouse and human intestinal adenomas. Collectively our results suggest that the previously identified feed-forward mechanisms between progastrin and Wnt signaling is a hallmark of early neoplastic transformation in mouse and human colonic adenomas., Competing Interests: Declaration of Competing Interest None of the authors has a conflict of interest, (Copyright © 2020. Published by Elsevier Inc.)

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (<5 cycles of 6 h each) did not alter antigenicity and allowed for detection of gene mutations, amplifications or even fusion transcripts. EDTA showed superiority for in situ hybridization techniques. According to these results and our institutional experience, we propose recommendations for decalcification of bone samples, from biopsies to surgical specimens.

Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I) 3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2 -silencing RNAs (siRNAs), -3554, -3825 and -4125, selected at the 3'-end of the murine Col1a2 transcript were tested in vitro and in vivo . In murine embryonic fibroblasts Col1a2- siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2- siRNA-3554 was proved also in vivo . Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2- siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Osteosarcoma (OS) is the main primary bone cancer, presenting poor prognosis and difficult treatment. An innovative therapy may be found in cold plasmas, which show anti-cancer effects related to the generation of reactive oxygen and nitrogen species in liquids. In vitro models are based on the effects of plasma-treated culture media on cell cultures. However, effects of plasma-activated saline solutions with clinical application have not yet been explored in OS. The aim of this study is to obtain mechanistic insights on the action of plasma-activated Ringer's saline (PAR) for OS therapy in cell and organotypic cultures. To that aim, cold atmospheric plasma jets were used to obtain PAR, which produced cytotoxic effects in human OS cells (SaOS-2, MG-63, and U2-OS), related to the increasing concentration of reactive oxygen and nitrogen species generated. Proof of selectivity was found in the sustained viability of hBM-MSCs with the same treatments. Organotypic cultures of murine OS confirmed the time-dependent cytotoxicity observed in 2D. Histological analysis showed a decrease in proliferating cells (lower Ki-67 expression). It is shown that the selectivity of PAR is highly dependent on the concentrations of reactive species, being the differential intracellular reactive oxygen species increase and DNA damage between OS cells and hBM-MSCs key mediators for cell apoptosis.

IL-34 is a proinflammatory cytokine implicated in rheumatoid arthritis (RA). The current study aimed to assess the IL-34 expression in response to two members of the transforming growth factor (TGF)-β family, TGF-β1 and bone morphogenetic protein (BMP)-2, in synovial fibroblasts from RA patients. IL-34, TGF-β1, and BMP-2 productions were measured in patient synovial fluids by enzyme-linked immunosorbent assay. IL-34 mRNA levels were quantified by real-time quantitative PCR in human synovial fibroblasts and murine mesenchymal stem cells. Pharmacologic inhibitions were used to determine the involvement of activin receptor-like kinase 1 (ALK1) and ALK5 downstream TGF-β1 and BMP-2. IL-34, TGF-β1, and BMP-2 were expressed in synovial fluids from RA patients. We found a significant correlation between IL-34 and TGF-β1 expressions. Levels of both IL-34 and TGF-β1 were thus correlated with the total leukocyte counts in the synovial fluids. TGF-β1 and BMP-2 decreased IL-34 expression in the synovial fibroblasts or in murine mesenchymal stem cells in a dose- and time-dependent manner through ALK5 and ALK1 pathways, respectively. In addition, TGF-β1 and BMP-2 antagonized tumor necrosis factor α-induced IL-34 gene expression. This work identifies TGF-β1 and BMP-2 as potent inhibitors of IL-34 expression in RA synovial fibroblasts. These cytokines, as upstream inhibitors of IL-34, may thus contribute to antagonize inflammation and bone erosions in RA., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Polyethylene micro-sized wear particles released from orthopedic implants promote inflammation and osteolysis; however, less is known about the bioactivity of polyethylene nanosized wear particles released from the last generation of polymer-bearing surfaces. We aim to assess the internalization of fluorescent polyethylene-like nanoparticles by both human macrophages and osteoclasts and also, to determine their effects in osteoclastogenesis in vitro. Human macrophages and osteoclasts were incubated with several ratios of fluorescent polyethylene-like nanoparticles between 1 and 72 h, and 4 h, 2, 4, 6, and 9 days, respectively. The internalization of nanoparticles was quantified by flow cytometry and followed by both confocal and video time-lapse microscopy. Osteoclast differentiation and activity was semiquantified by tartrate-resistant acid phosphatase (TRAP) staining, TRAP mRNA relative expression, and pit resorption assay, respectively. Macrophages, osteoclast precursors and mature osteoclasts internalized nanoparticles in a dose- and time-dependent manner and maintained their resorptive activity. In addition, nanoparticles significantly increased the osteoclastogenesis as shown by upregulation of the TRAP expressing cell number. We conclude that polyethylene-like nanosized wear particles promote osteoclast differentiation without alteration of bone resorptive activity of mature osteoclasts and they could be considered as important actors in periprosthetic osteolysis of the last new generation of polymer-bearing surfaces. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2649-2657, 2016., (© 2016 Wiley Periodicals, Inc.)

Interleukin (IL)-36α, IL-36β and IL-36γ are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL-36Ra or IL-38, another potential IL-36 inhibitor, limit uncontrolled inflammation. The expression and role of IL-36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod-induced mouse skin inflammation and in human psoriasis, expression of IL-36α, γ and IL-36Ra, but not IL-36β and IL-38 mRNA, was induced and correlated with IL-1β and T helper type 17 (Th17) cytokines (IL-17A, IL-22, IL-23, CCL20). In mice with collagen-induced arthritis and in the synovium of patients with RA, IL-36α, β, γ, IL-36Ra and IL-38 were all elevated and correlated with IL-1β, CCL3, CCL4 and macrophage colony-stimulating factor (M-CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium-induced colitis and in patients with CD, only IL-36α, γ and IL-38 were induced at relatively low levels and correlated with IL-1β and IL-17A. We suggest that only a minor subgroup of patients with RA (17-29%) or CD (25%) had an elevated IL-36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL-36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68⁺ macrophages, dendritic/Langerhans cells and CD79α⁺ plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL-36β and IL-36Ra were produced constitutively, but IL-36α, γ and IL-38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL-36 agonists/antagonists ratio., (© 2016 British Society for Immunology.)

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.

Strongly solvatochromic fluorophores are devised, containing alkyl chains and enable to self-assemble as very bright fluorescent organic nanoparticles (FONs) in water (Φf = 0.28). The alkyl chains impart each fluorophore with strongly hydrophobic surroundings, causing distinct emission colors between FONs where the fluorophores are associated, and their disassembled state. Such color change is harnessed to assess the long-term fate of FONs in both cancer cells and monocytes/macrophages. Disintegration of the orange-emitting FONs by monocytes/macrophages is evidenced through the formation of micrometer green-yellowish emitting vesicles. By contrast, cancer cells retain longer the integrity of organic nanoparticles. In both cases, no significant toxicity is detected, making FONs as valuable bioimaging agents for cell tracking with weak risks of deleterious accumulation and low degradation rate., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Interleukin-34 (IL-34) is a newly-discovered homodimeric cytokine that regulates, like Macrophage Colony-Stimulating Factor (M-CSF), the differentiation of the myeloid lineage through M-CSF receptor (M-CSFR) signaling pathways. To date, both cytokines have been considered as competitive cytokines with regard to the M-CSFR. The aim of the present work was to study the functional relationships of these cytokines on cells expressing the M-CSFR. We demonstrate that simultaneous addition of M-CSF and IL-34 led to a specific activation pattern on the M-CSFR, with higher phosphorylation of the tyrosine residues at low concentrations. Similarly, both cytokines showed an additive effect on cellular proliferation or viability. In addition, BIAcore experiments demonstrated that M-CSF binds to IL-34, and molecular docking studies predicted the formation of a heteromeric M-CSF/IL-34 cytokine. A proximity ligation assay confirmed this interaction between the cytokines. Finally, co-expression of the M-CSFR and its ligands differentially regulated M-CSFR trafficking into the cell. This study establishes a new foundation for the understanding of the functional relationship between IL-34 and M-CSF, and gives a new vision for the development of therapeutic approaches targeting the IL-34/M-CSF/M-CSFR axis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII), but little is known about the pathogenicity of this species in such infections that are increasing due to the ageing of the population. We report how this pathogen interacts with human osteoblastic MG-63 cells in vitro, by comparing 20 OII E. coli strains to two Staphylococcus aureus and two Pseudomonas aeruginosa strains. LDH release assay revealed that 6/20 (30%) OII E. coli induced MG-63 cell lysis whereas none of the four control strains was cytotoxic after 4 h of coculture. This high cytotoxicity was associated with hemolytic properties and linked to hlyA gene expression. We further showed by gentamicin protection assay and confocal microscopy that the non-cytotoxic E. coli were not able to invade MG-63 cells unlike S. aureus strains (internalization rate <0.01% for the non-cytotoxic E. coli versus 8.88 ± 2.31% and 4.60 ± 0.42% for both S. aureus). The non-cytotoxic E. coli also demonstrated low adherence rates (<7%), the most adherent E. coli eliciting higher IL-6 and TNF-α mRNA expression in the osteoblastic cells. Either highly cytotoxic or slightly invasive OII E. coli do not show the same infection strategies as S. aureus towards osteoblasts., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cell types of the intestinal epithelium and describe the first marker signature allowing their unambiguous identification. We demonstrate that although mature tuft cells express DCLK1, a putative marker of quiescent stem cells, they are post-mitotic, short lived, derive from Lgr5-expressing epithelial stem cells, and are found in mouse and human tumors. We show that whereas the ATOH1/MATH1 transcription factor is essential for their differentiation, Neurog3, SOX9, GFI1, and SPDEF are dispensable, which distinguishes these cells from enteroendocrine, Paneth, and goblet cells, and raises from three to four the number of secretory cell types in the intestinal epithelium. Moreover, we show that tuft cells are the main source of endogenous intestinal opioids and are the only epithelial cells that express cyclooxygenase enzymes, suggesting important roles for these cells in the intestinal epithelium physiopathology.

The hepatitis C virus genome encodes a polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases to yield 10 mature protein products (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Although most cleavages in hepatitis C virus polyprotein precursor proceed to completion during or immediately after translation, the cleavages mediated by a host cell signal peptidase are partial at the E2/p7 and p7/NS2 sites, leading to the production of an E2p7NS2 precursor. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can function as signal peptides. When fused to a reporter protein, the signal peptides of p7 and NS2 were efficiently cleaved. However, when full-length p7 was fused to the reporter protein, partial cleavage was observed, indicating that a sequence located N-terminally of the signal peptide reduces the efficiency of p7/NS2 cleavage. Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both the E2/p7 and p7/NS2 sites. Finally, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane alpha-helix within the P' region might impose additional structural constraints to the cleavage sites. The insertion of a linker polypeptide sequence between P-3' and P-4' of the cleavage site released these constraints and led to improved cleavage efficiency. Such constraints in the processing of a polyprotein precursor are likely essential for hepatitis C virus to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.