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Dental implants are one of the most frequently used treatment options for tooth replacement. Approximately 30% of patients with dental implants develop peri-implantitis, which is an oral inflammatory disease that leads to loss of the supporting tissues, predominately the bone. For the development of future therapeutic strategies, it is essential to understand the molecular pathophysiology of human dental peri-implant infections. Here, we describe the gene and protein expression patterns of peri-implantitis bone tissue compared with healthy peri-implant bone tissue. Furthermore, cells from the osteoblastic lineage derived from peri-implantitis samples were immortalized and characterized. We applied microarray, quantitative reverse transcription polymerase chain reaction, fluorescence-activated cell sorting, and Western blot analyses. The levels of typical bone matrix molecules, including SPP1, BGLAP, and COL9A1, in patients with peri-implantitis were reduced, while the inflammation marker interleukin 8 (IL8) was highly expressed. RUNX2, one of the transcription factors of mature osteoblasts, was also decreased in peri-implantitis. Finally, the human telomerase reverse transcriptase immortalized cell line from peri-implantitis exhibited a more fibro-osteoblastic character than did the healthy control. [ABSTRACT FROM PUBLISHER]

Components of the insulin-like growth factor (IGF) system are involved in orofacial physiology, development and malformation. Recently, we could localize components of the IGF system in oral and dental human and rodent tissues. IGF-2 and its high affinity binding protein, IGFBP-6, are important for developmental processes, where they regulate e.g. proliferation. IGFBP-6 has an inhibitory effect on IGF-II. While we already could show the expression of these factors in the adult periodontal ligament, no data concerning their appearance and distribution during human odontogenesis and jaw development were available. Serial sections of decalcified and paraffin-embedded specimens from the jaw regions of human embryos (6th - 22nd gestational week (gw)) were investigated immunohistochemically using mono- and polyclonal antibodies against IGF-2 and IGFBP-6. The results mainly showed a colocalization and an increasing intensity of immunoreactivity with gestational age. Immunostaining could already be demonstrated in the dental lamina and later in the enamelum organ. Strong staining appeared in the enamelum knot and Hertwig's epithelial sheeth (HERS). With the beginning of the 10th gw both factors could be localized in dental papilla and follicle. While Meckel's cartilage was unstained, strong reactivity could be seen in the jaw blastema and in osteoblasts. The results show that IGF-2 and IGFBP-6 are both involved in odontogenesis and orofacial development. Findings in the enamelum knot and HERS indicate a role of IGF-2 in proliferation. Its appearance in the developing jaw bones reveals a function in osteogenesis. The colocalization with IGFBP-6 demonstrates biological interaction with its ligand, thus probably controlling mitogenesis. [ABSTRACT FROM AUTHOR]

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 μm s -1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 μm s -1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis , suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

Background: Changes in the proteome of oral cells during periodontitis have rarely been investigated. This lack of information is partially attributed to the lack of human cell lines derived from the oral cavity for in vitro research. The objective of the present study was to create cell lines from relevant oral tissues and compare protein expression in cells cultured alone and in cells co-cultivated with periodontitis-associated bacterial strains., Methods: We established human cell lines of gingival keratinocytes, osteoblastic lineage cells from the alveolar bone, periodontal ligament fibroblasts, and cementum cells. Using state-of-the-art label-free mass spectrometry, we investigated changes in the proteomes of these cells after co-cultivation with Aggregatibacter actinomycetemcomitans and Eikenella corrodens for 48 h., Results: Gingival keratinocytes, representing ectodermal cells, exhibited decreased expression of specific keratins, basement membrane components, and cell-cell contact proteins after cultivation with the bacterial strains. Mesodermal lineage cells generally exhibited similar proteomes after co-cultivation with bacteria; in particular, collagens and integrins were expressed at higher levels., Conclusions: The results of the present study will help us elucidate the cellular mechanisms of periodontitis. Although co-cultivation with two periodontitis-associated bacterial strains significantly altered the proteomes of oral cells, future research is needed to examine the effects of complex biofilms mimicking in vivo conditions.

Objective: This study investigated the effect of eluates of conventional and 3D-printed resin materials for manufacturing temporary dental restorations on gingival keratinocytes., Methods: Three-dimensional (3D)-printed resin materials: 3Delta temp (Deltamed), NextDent MFH (Nextdent), Freeprint temp (Detax), GC temp (GC), were compared to Grandio disc (Voco) and Luxatemp (DMG). Human gingival keratinocytes (IHGKs) were exposed to eluates of the materials and XTT assays were performed at 24 h, 48 h, 72 h, or 144 h. For quantification of the proinflammatory response, the protein amount of IL-6 and 8 was determined in the supernatants using ELISA. One-way ANOVA with post hoc analysis was used to compare differences in cell viability and IL-6 and IL-8 levels between groups., Results: At 24 h, and more remarkably at 48 h, a significant decrease in cell viability occurred for the 3D-printed materials compared to the untreated IHGKs, but also compared to Grandio disc and Luxatemp. Except for the expression of IL-8 in presence of the eluate of Grandio disc at 24 and 48 h, all tested materials caused attenuation of IL-6 and 8 from IHGKs for any observation period., Conclusions: The materials for additive manufacturing affect cell proliferation differently than the subtractive manufactured material Grandio disc and the conventional material Luxatemp., Clinical Significance: In comparison to conventional and subtractive manufactured restorations, 3D printed temporary restorations might induce more negative effects on the gingival and probably also on pulpal health since viability and the proinflammatory response of oral keratinocytes are more intensively affected by these materials., (© 2022 The Authors. Journal of Esthetic and Restorative Dentistry published by Wiley Periodicals LLC.)

Objective: The aim of this study was to investigate the roles of SMURF1 and SMURF2 in progenitor cells from the human knee in late-stage osteoarthritis (OA)., Design: We applied immunohistochemistry, immunocytochemistry, RNAi, lentiviral transfection, and Western blot analysis. We obtained chondrogenic progenitor cells (CPCs) from the articular cartilage and meniscus progenitor cells (MPCs) from the nonvascularized part of the meniscus., Results: SMURF1 and SMURF2 appeared in both osteoarthritic tissues. CPCs and MPCs exhibited comparable amounts of these proteins, which influence the balance between RUNX2 and SOX9. The overexpression of SMURF1 reduced the levels of RUNX2, SOX9, and TGFBR1. The overexpression of SMURF2 also reduced the levels of RUNX2 and TGFBR1, while SOX9 levels were not affected. The knockdown of SMURF1 had no effect on RUNX2, SOX9, or TGFBR1. The knockdown of SMURF2 enhanced RUNX2 and SOX9 levels in CPCs. The respective protein levels in MPCs were not affected., Conclusions: This study shows that SMURF1 and SMURF2 are regulatory players for the expression of the major regulator transcription factors RUNX2 and SOX9 in CPCs and MPCs. Our novel findings may help elucidate new treatment strategies for cartilage regeneration.

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca 2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration., (© 2021 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Objective: In osteoarthritis (OA), a loss of healthy cartilage extracellular matrix (ECM) results in cartilage degeneration. Attracting chondrogenic progenitor cells (CPCs) to injury sites and stimulating them toward chondrogenic expression profiles is a regenerative approach in OA therapy. High mobility group box 1 protein (HMGB1) is associated with chemoattractant and proinflammatory effects in various pathological processes. Here, we investigate the migratory effects of HMGB1 in knee OA and CPCs for the first time., Design: Immunohistochemistry, immunoblotting, and immunocytochemistry were performed to identify HMGB1 and its receptors, receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4) in OA knee tissue, chondrocytes, and CPCs. In situ hybridization for HMGB1 mRNA was performed in CPCs ex vivo . The chemoattractant effects of HMGB1 on CPCs were analyzed in cell migration assays., Results: HMGB1 expression in OA tissue and OA chondrocytes was higher than in healthy specimens and cells. HMGB1, RAGE, and TLR4 were expressed in CPCs and chondrocytes. In situ hybridization revealed HMGB1 mRNA in CPCs after migration into OA knee tissue, and immunohistochemistry confirmed HMGB1 expression at the protein level. Stimulation via HMGB1 significantly increased the migration of CPCs., Conclusions: Our results show the chemoattractant role of HMGB1 in knee OA. HMGB1 is released by chondrocytes and has migratory effects on CPCs. These effects might be mediated via RAGE and TLR4. The in vitro and ex vivo results of this study need to be confirmed in vivo .

Osteoarthritis (OA) is the most prevalent chronic joint disease that affects a large proportion of the elderly population. Chondrogenic progenitor cells (CPCs) reside in late-stage OA cartilage tissue, producing a fibrocartilaginous extracellular matrix; these cells can be manipulated in vitro to deposit proteins of healthy articular cartilage. CPCs are under the control of SOX9 and RUNX2. In our earlier studies, we showed that a knockdown of RUNX2 enhanced the chondrogenic potential of CPCs. Here we demonstrate that CPCs carrying a knockout of RAB5C, a protein involved in endosomal trafficking, exhibited elevated expression of multiple chondrogenic markers, including the SOX trio, and increased COL2 deposition, whereas no changes in COL1 deposition were observed. We report RAB5C as an attractive target for future therapeutic approaches designed to increase the COL2 content in the diseased joint., Competing Interests: The authors have no potential conflicts of interest to declare with respect to the research, authorship, and/or publication of this article. Inclusion and Diversity statement: We worked to ensure gender balance and ethnic or other types of diversity in the recruitment of human subjects. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community., (© 2021.)

Objective: This study evaluated the gene expression and protein synthesis of sex hormone receptors in human periodontal ligament cells (PDLCs) in relation to donor- and tooth-specific factors with the aim to clarify the debate about sex hormone receptors in PDLCs., Design: The expression patterns of estrogen receptors (genes: ESR1 and ESR2; proteins: ERα and ERβ), androgen receptor (AR) and progesterone receptor (PR) were investigated in the context of immortalization status, previous orthodontic tooth movement (OTM), donor age, sex and hormonal stimulation in PDLCs from 14 healthy donors (male: n = 8, female: n = 6; adolescents: n = 8, adults: n = 6) using quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry., Results: For ERβ, the full-length isoform ERβ1 and truncated variants were detected. For ERα, the expected isoform ERα66 was not observed, but a novel isoform ERα36 was detected. Immortalization status, previous OTM and donor age had no impact on ESR1 and ESR2 expression. Estradiol stimulation for 24 h doubled the ratio of ESR2/ESR1 in PDLCs from female but not male donors, indicating sex-specific patterns of receptor expression. AR and PR demonstrated insufficient protein synthesis in PDLCs., Conclusions: The data revealed a pivotal role for and complex interplay between ERα and ERβ in human PDLCs regardless of variable donor characteristics. Therefore, PDLC biology might be altered in patients of each age group and both sexes due to hormonal changes. This should be kept in mind during periodontic and orthodontic treatment of patients with special hormonal status., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of the study was to clarify whether orthodontic forces and periodontitis interact with respect to the anti-apoptotic molecules superoxide dismutase 2 (SOD2) and baculoviral IAP repeat-containing protein 3 (BIRC3). SOD2, BIRC3, and the apoptotic markers caspases 3 (CASP3) and 9 (CASP9) were analyzed in gingiva from periodontally healthy and periodontitis subjects by real-time PCR and immunohistochemistry. SOD2 and BIRC3 were also studied in gingiva from rats with experimental periodontitis and/or orthodontic tooth movement. Additionally, SOD2 and BIRC3 levels were examined in human periodontal fibroblasts incubated with Fusobacterium nucleatum and/or subjected to mechanical forces. Gingiva from periodontitis patients showed significantly higher SOD2, BIRC3, CASP3, and CASP9 levels than periodontally healthy gingiva. SOD2 and BIRC3 expressions were also significantly increased in the gingiva from rats with experimental periodontitis, but the upregulation of both molecules was significantly diminished in the concomitant presence of orthodontic tooth movement. In vitro, SOD2 and BIRC3 levels were significantly increased by F. nucleatum , but this stimulatory effect was also significantly inhibited by mechanical forces. Our study suggests that SOD2 and BIRC3 are produced in periodontal infection as a protective mechanism against exaggerated apoptosis. In the concomitant presence of orthodontic forces, this protective anti-apoptotic mechanism may get lost.

The human urea transporter SLC14A1 (HUT11/UT-B) has been suggested as a marker for the adipogenic differentiation of bone cells with a relevance for bone diseases. We investigated the function of SLC14A1 in different cells models from bone environment. SLC14A1 expression and cytokine production was investigated in bone cells obtained from patients with osteoporosis. Gene and protein expression of SLC14A1 was studied during adipogenic or osteogenic differentiation of human mesenchymal progenitor cells (hMSCs) and of the single-cell-derived hMSC line (SCP-1), as well as in osteoclasts and chondrocytes. Localization was determined by histochemical methods and functionality by urea transport experiments. Expression of SLC14A1 mRNA was lower in cells from patients with osteoporosis that produced high levels of cytokines. Accordingly, when adding a combination of cytokines to SCP-1 SLC14A1 mRNA expression decreased. SLC14A1 mRNA expression decreased after both osteogenic and more pronounced adipogenic stimulation of hMSCs and SCP-1 cells. The highest SLC14A1 expression was determined in undifferentiated cells, lowest in chondrocytes and osteoclasts. Downregulation of SLC14A1 by siRNA resulted in an increased expression of interleukin-6 and interleukin-1 beta as well as adipogenic markers. Urea influx through SLC14A1 increased expression of osteogenic markers, adipogenic markers were suppressed. SLC14A1 protein was localized in the cell membrane and the cytoplasm. Summarizing, the SLC14A1 urea transporter affects early differentiation of hMSCs by diminishing osteogenesis or by favoring adipogenesis, depending on its expression level. Therefore, SLC14A1 is not unequivocally an adipogenic marker in bone. Our findings suggest an involvement of SLC14A1 in bone metabolism and inflammatory processes and disease-dependent influences on its expression., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

The complement of cell surface proteins, collectively referred to as the surfaceome, is a useful indicator of normal differentiation processes, and the development of pathologies such as osteoarthritis (OA). We employed biochemical and proteomic tools to explore the surfaceome and to define biomarkers in chondrogenic progenitor cells (CPC) derived from human OA knee articular cartilage. These cells have great therapeutic potential, but their unexplored biology limits their clinical application. We performed biotinylation combined with glycocapture and high throughput shotgun proteomics to define the surface proteome of human bone marrow mesenchymal stem cells (MSCs) and human CPCs. We prepared cell surface protein-enriched fractions from MSCs and CPCs, and then a proteomic approach was used to compare and evaluate protein changes between undifferentiated MSCs and CPCs. 1256 proteins were identified in the study, of which 791 (63%) were plasma membrane, cell surface or extracellular matrix proteins. Proteins constituting the surfaceome were annotated and categorized. Our results provide, for the first time, a repository of quantitative proteomic data on the surfaceome of two closely related cell types relevant to cartilage biology and OA. These results may provide novel insights into the transformation of the surfaceome during chondrogenic differentiation and phenotypic changes during OA development.

Osteoarthritis (OA) is the most common chronic joint disease and leads to the degradation of the extracellular matrix by an imbalance between anabolic and catabolic processes. TGF-β3 (transforming growth factor beta-3) and epidermal growth factor (EGF) influence the osteochondrogenic potential of chondrocytes. In this study, we compared the expression of mediators and receptors in the TGF-β3 and EGF pathways, as well as biglycan (BGN), in healthy and diseased chondrocytes. Furthermore, we used chondrogenic progenitor cells (CPCs) for in vitro stimulation and knockdown experiments to elucidate the effects of TGF-β3 and EGF on the chondrogenic potential. Our results demonstrate that the expression of TGF-beta receptor type-1 (TGFBRI) and epidermal growth factor receptor (EGFR) is altered in diseased chondrocytes as well as in CPCs. Moreover, TGF-β3 and EGF stimulation influenced the expression levels of BGN, SRY (sex determining region Y)-box 9 (SOX9), and Runt-related transcription factor 2 (RUNX2) in CPCs. Therefore, changes in TGFBRI and EGFR expression likely contribute to the degenerative and regenerative effects seen in late stages of OA.