Modulation of osteoarthritis-related microRNAs using locked nucleic acid-antisense oligonucleotides boosts chondrogenic lineage commitment of mesenchymal progenitors

MicroRNAs (miRNAs) are small non-coding double-stranded RNA with sizes of 20-25 nucleotides, and inhibit protein translation by binding the 3'-untranslated region of target mRNA. Each miRNA can regulate multiple mRNAs and each mRNA can be targeted by a number of miRNAs. In cancer, miRNAs can act as not only tumor suppressor genes but also oncogenes (OncomiR). Most recent study has demonstrated OncomiR addiction in mouse pre-B-cell lymphoma. OncomiR addiction may also provide therapeutic opportunities in human cancers such as oncogene addiction. In this study, we have attempted to identify an OncomiR in human oral cancer cells through functional screening and considered whether targeting miRNA can be possible for cancer therapy. First, we performed functional screening for OncomiR in human oral cancer cells by the use of miRCURY LNATM microRNA Knockdown Library (Exiqon). We transfected 918 locked nucleic acid (LNA) antisense oligonucleotides for specific human mature miRNAs into human oral squamous cell carcinoma cells (GFP-SAS) and salivary gland cancer cells (GFP-ACCM). After transfection for 80 hours, each cell growth was evaluated. LNA antisense oligonucleotides against microRNA-361-3p (LNA-miR-361-3p) showed a remarkable growth inhibition in both types of cells as compared with non-targeting LNA oligonucleotides. We also observed the change of cell morphology, diminution of colony size, and a number of non-adherent cells after transfection of LNA-miR-361-3p. Subsequently, we examined the knockdown effect of LNA-miR-361-3p in GFP-SAS cells by quantitative RT-PCR. Compared with control oligonucleotides, the expression of miR-361-3p was significantly reduced by 71%. These effects of LNA-miR-361-3p were not observed by transfection of DNA or RNA antisense oligonucleotides for miR-361-3p. Next, we transfected synthetic human mature miR-361-3p into GFP-SAS cells to investigate the effect of miR-361-3p overexpression. Cell growth resulted in a significant 20% increase compared with non-targeting control miRNA. Furthermore, co-transfection of LNA-miR-361-3p and its decoy oligonucleotides abrogated the growth inhibitory effect by LNA-miR-361-3p in GFP-SAS cells. These results suggest that miR-361-3p functions as an OncomiR in human oral cancer cells and LNA antisense oligonucleotides are useful and efficient for silencing miRNA. Targeting miR-361-3p with LNA antisense oligonucleotides may be a useful therapeutic approach for patients with oral cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 145. doi:10.1158/1538-7445.AM2011-145

Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

The aim of this study was to explore the role of microRNA-143-3p (miR-143-3p) in cartilage injury, and to investigate the possible underlying mechanism. A chondrogenic differentiation cell model was established in bone marrow mesenchymal stem cells (BMSCs). The mRNA expression levels of runt-related transcription factor 2 (RUNX2), miR-143-3p and bone morphogenetic protein 2 (BMPR2) in BMSCs were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) after 0 d, 5 d and 10 d, respectively. Mesenchymal stem cells (MSCs) were transfected with miR-143-3p mimics and its control in accordance with the liposome method. Alcian blue colorimetric assay was used to evaluate proteoglycan deposition of MSCs. Meanwhile, qRT-PCR and Western blot were performed to analyze the expression levels of ACAN and COL2A1. Luciferase reporter gene assay was applied to verify the binding status of miR-143-3p and BMPR2 3'UTR. Also, proteoglycan deposition and the expression of ACAN and COL2A1 were detected after simultaneous transfection of miR-143-3p mimics and BMPR2 overexpression plasmid. 0 d, 5 d and 10 d after inducing cartilage differentiation, the mRNA expression levels of RUNX2 and BMPR2 were markedly increased. However, the expression level of miR-143-3p was significantly decreased with the prolongation of induction period. After transfection with miR-143-3p mimics, the level of miR-143-3p in MSCs was remarkably increased. Alcian blue colorimetric assay and staining assay showed that the deposition of proteoglycans in the mimics group was significantly lower than that of the control group. Meanwhile, after overexpressing miR-143-3p, the levels of cartilage differentiation marker proteins including ACAN and COL2A1 were remarkably reduced. Luciferase report gene assay indicated that miR-143-3p could negatively regulate BMPR2 by binding to its 3'UTR. In addition, overexpression of BMPR2 could strikingly reverse the above effects of overexpressed miR-143-3p. During chondrogenic differentiation, the level of miR-143-3p was decreased. Moreover, miR-143-3p could regulate the differentiation process by targeting BMPR2 in BMSCs.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells

Osteoarthritis (OA) is a chronic joint disease characterized by a progressive and irreversible degeneration of articular cartilage. Among the environmental risk factors of OA, tobacco consumption features prominently, although, there is a great controversy regarding the role of tobacco smoking in OA development. Among the numerous chemicals present in cigarette smoke, nicotine is one of the most physiologically active molecules. The aim of the study was (i) to measure the impact of nicotine on the proliferation and chondrogenic differentiation of mesenchymal stem cells from the human Wharton's jelly (hWJ-MSCs) into chondrocytes, (ii) to investigate whether the α7 nicotinic acetylcholine receptors (nAChRs) was expressed in hWJ-MSCs and could play a role in the process. The project benefits from the availability of an umbilical cord bank from which hWJ-MSCs were originated. The hWJ-MSCs were cultured and used up to passage 5. The proliferation of hWJ-MSCs with 5μM nicotine was measured by the MTT assay on the 1st, 2nd, 3rd, and 6th day. Flow cytometry analysis was used to detect cell apoptosis/necrosis by Annexin V/PI double-staining. The chondrogenic differentiation grade of hWJ-MSCs induced by TGFβ3 was assessed by the Sirius red and Alcian blue staining. The expression of markers genes was followed by quantitative real-time PCR. The expression of nAChRs was followed by RT-PCR. The functional activity of α7 nAChR was evaluated by calcium (Ca2+) influx mediated by nicotine using the Fluo-4 NW Calcium assay. The proliferation of hWJ-MSCs was significantly impaired by nicotine (5μM) from the 3rd day of treatment, but nicotine did not significantly induce modifications on the viability of hWJ-MSCs. Alcian blue staining indicated that the amount of proteoglycan was more abundant in control group than in the nicotine group, but no difference was observed on the total collagen amount using Sirius red staining. The mRNA expression of Sox9, type II collagen (Col2a1), aggrecan in control group was higher than in the nicotine group. We found that hWJ-MSCs expressed α7 nAChR. The receptor agonist nicotine caused calcium (Ca2+) influx into hWJ-MSCs suggesting that the calcium ion channel α7 homopolymer could mediate this response. At the concentration used, nicotine had an adverse effect on the proliferation and chondrogenic differentiation of hWJ-MSCs which was probably impaired through a α7 nAChR mediation.

The aim of the study was to explore the role of microRNA-23c in the differentiation of marrow stromal cells (MSCs) to chondrocytes and its potential mechanism. MSCs were first isolated from rat bone marrow for cell culture. Surface antigens of MSCs (CD29 and CD34) were identified by flow cytometry. MSCs were induced for chondrogenic differentiation in MCDM (Mesenchymal Stem Cell Chondrogenic Differentiation Medium) for 0, 3, and 7 days, respectively, followed by detection of RUNX2, microRNA-23c and FGF2 expressions by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Alcian blue staining was performed to access proteoglycan deposition in MSCs transfected with microRNA-23c mimics or inhibitor. Western blot was conducted to detect the protein expressions of ACAN and COL2A1 in MSCs. The binding condition between microRNA-23c and FGF2 was verified by dual-luciferase reporter gene assay. Finally, MSCs were co-transfected with microRNA-23c mimics and FGF2 overexpression plasmid for rescue experiments. On the fourth day of MSCs isolation, MSCs were in an elongated shape. Flow cytometry results showed positive expression of CD29 and negative expression of CD34, which were consistent with MSCs phenotype. QRT-PCR data elucidated that the mRNA levels of RUNX2 and FGF2 gradually increased, whereas microRNA-23c expression decreased with the prolongation of chondrogenic differentiation. Transfection of microRNA-23c mimics in MSCs remarkably elevated microRNA-23c expression. Alcian blue staining showed that microRNA-23c overexpression results in less proteoglycan deposition in MSCs than that of controls. Both mRNA and protein expressions of ACAN and COL2A1 decreased after microRNA-23c overexpression. Dual-luciferase reporter gene assay confirmed that FGF2 binds to microRNA-23c. Further Western blot results demonstrated that FGF2 expression is negatively regulated by microRNA-23c. FGF2 overexpression reversed the inhibitory effects of microRNA-23c on proteoglycan deposition, as well as expressions of ACAN and COL2A1. MicroRNA-23c expression decreases during chondrogenic differentiation of MSCs, which inhibits MSCs differentiation to chondrocytes by inhibiting FGF2.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

CORR Synthesis: What Is the Evidence for the Clinical Use of Stem Cell-based Therapy in the Treatment of Osteoarthritis of the Knee?

The overall goal of my PhD studies was to analyze how the regulation of angiogenesis, a key factor that plays very important roles in tissue repair, could ultimately influence cartilage formation by mesenchymal stromal cells (MSC) and by differentiated nasal chondrocytes (NC) as a proof of concept. Due to their multipotency and great self-renewal capacity, MSC is a highly attractive cell source for cartilage formation and regeneration. MSC have immunomodulatory properties and are in direct contact with inflammatory cells (monocytes) in the cartilage repair environment. By directly regulating the inflammatory response of monocytes and by regulating their phenotype, MSC could significantly benefit from their ability to control inflammatory events during cartilage repair processes. Thus, we also assessed whether MSC could modulate the properties and the phenotype of monocytes in order for monocytes to actively aid MSC in cartilage repair. In chapter 1, we sought a proof of principle to confirm that the blocking of angiogenesis does indeed improve cartilage formation when using genetically-modified nasal chondrocytes (NCs) or antiangiogenic peptides associated with NCs. For this purpose, NCs were genetically-modified to express mouse soluble VEGF receptor-2 (sFlk-1) or were associated with an antiangiogenic peptide in order to have their chondrogenic capacity assessed in vitro and in vivo. Improved cartilage regeneration could be observed after in vivo implantation of NCs in an ectopic nude mouse model. Whereas the anti-angiogenic approaches did not improve chondrogenesis in vitro, frank chondrogenesis occured in vivo only in the constructs generated by NCs expressing sFlk-1 or treated with the peptide. Blood vessel ingrowth was significantly hampered in the anti-angiogenic experimental groups when compared with naive NCs, which correlated with chondrogenis improvement. Strikingly, the anti-angiogenic effect was even more evident when NC donors with low chondrogenic capacity were used, and no-preculture with chondrogenic soluble factors was performed prior to in vitro implantation of the constructs. In chapter 2, we investigated how angiogenesis control by genetically-modified bone marrow-derived MSC could augment their chondrogenic potential in vivo. These MSC were genetically modified to release sFlk-1, the soluble version of VEGF receptor 2 (VEGFR2), which acted as a decoy receptor and could sequester VEGF from the immediate surroundings of MSC. Importantly, no external morphogens were supplemented in order for MSC chondrogenic differentiation to occur. Moreover, the in vivo chondrogenic capacity of sFlk-1-releasing MSC was assessed up to 12 weeks by an ectopic nude mouse model, in which collagen sponges seeded with MSC were implanted subcutaneously. Angiogenesis, as analyzed by blood vessel invasion, was markedly reduced in the constructs seeded with sFlk-1-releasing MSC. Frank and stable cartilage formation was only achieved once VEGF was blocked. In chapter 3, we aimed at investigating whether MSC can instruct monocytes to acquire traits of mesenchymal progenitors or tissue repair macrophages when in direct or indirect contact with the latter. Thus, MSC could instruct monocytes to assist in the tissue-repairing process. MSC and monocytes were cocultured either in 3D collagen sponges or in transwells for a period of five consecutive days. We demonstrated that MSC instruct monocytes into acquiring a hybrid macrophage-mesenchymal phenotype, which varies greatly depending on which kind of contact with MSC the monocytes were exposed to (whether direct cell-to-cell contact or through the release of soluble factors by MSC). However, future studies will be needed to elucidate the mechanism in which MSC could instruct monocytes into differentiating into this hybrid state, and how one could better control this process in order for monocytes to optimally aid MSC in cartilage repair.

Objective To determine the chondrogenic potential of mesenchymal stem cells (BMSCs) under exposure of extremely low frequency electromagnetic fields (ELF-EMF) and discuss the application prospect of EMF in cartilage tissue engineering.Methods Cell pellets of rat bone marrow-derived BMSCs were cultured in vitro under the addition of fibroblast growth factor 2 (FGF-2) and transforming growth factor-β3 (TGF-β3).Pellet cultures were exposed to sinusoidal ELF-EMF (1.0 mT,50 Hz).After 3 weeks of inductive differentiation culture,chondrogenesis was detected by alcian blue staining,and quantitative real-time polymerase chain reaction (real-time PCR) was used for detection of chondrogenesis related proteins.Glycosaminoglycan (GAG) contents of pellet cultures were determined using the dimethylmethylene blue (DMMB) dye-binding assay.Results With the effects of growth factors,EMF could significantly promote chondrogenic differentiation of BMSCs pellet cultures,and the gene expression of collagen type Ⅱ,X and aggrecan in rat BMSCs was also significantly up-regulated.The gene expression of sex determining region Y box 2 (SOX9) did not show significant difference between EMF exposed groups and nonexposed groups.The glycosaminoglycan (GAG)/DNA ratio of EMF exposed pellet cultures reached 3.108 ±0.341,which was significantly higer than those unexposed pellet cultures.Conclusion Increased expression of collagen type Ⅱ,X and GAG content in pellet cultures under EMF exposure may contribute to the chonderogenic differentiation of rat BMSCs.EMF was able to stimulate and maintain chondrogenic differentiation of rat BMSCs under influence of growth factors.EMF alone,however,could not induce chondrogenic differentiation. Key words: Electromagnetic fields; Mesenchymal stem cells; Pellet culture; Chondrogenic differentiation

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single-cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated toward osteoblast lineage cells but also expressed higher levels of osteogenic-related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases. Editor's evaluation Data from scRNA-Seq analysis demonstrated that acute inflammation stimulates periodontal stem cells to differentiate into osteoblast lineage cells to protect the alveolar bone. In murine models and patients with apical periodontitis, the genes and proteins associated with osteogenesis were enriched. The studies help us understand how MSCs respond to inflammation during apical periodontitis disease progression. https://doi.org/10.7554/eLife.82537.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Oral diseases, particularly dental caries and periodontal diseases, affect 3.5 billion people worldwide (Disease et al., 2018). Untreated dental caries can directly lead to pulp necrosis and periapical lesions, resulting in apical periodontitis (AP). Individuals with at least one tooth affected by AP comprise up to 52% of cases, indicating that AP is a highly prevalent disease (Tibúrcio-Machado et al., 2021). Inflammation in the oral cavity can lead to destruction of surrounding periapical tissues and resorption of hard tissues, a consequence of the unbalanced interaction between infection and the immune response (Gazivoda et al., 2009; Márton and Kiss, 2014). Restoring and regenerating the destroyed periapical alveolar bone structures have always been a challenging task in clinical practice. Active inflammation, the tissue injury and the protective process all occur simultaneously in the setting of chronic AP (Márton and Kiss, 2014). Importantly, there is a complex assemblage of immune cell types involved in the pathogenesis, highlighting the importance of polymorphonuclear leukocytes, lymphocytes, and monocyte/macrophages in periapical defense (Braz-Silva et al., 2019; Nair, 2004). Notably, an increasing number of studies report the involvement of mesenchymal stem cells (MSCs) in the protective action that occurs during oral inflammatory diseases, whereby MSCs exert immunomodulatory effects and have regenerative potential (Li et al., 2014; Márton and Kiss, 2000; Nair, 2004). MSC markers such as CD44, CD73, CD90, CD106, and STRO-1 have been observed in human periapical inflammatory tissues (Estrela et al., 2019; Liao et al., 2011). Cells isolated from the inflamed periapical region were able to produce colony-forming unit-fibroblasts (CFU-Fs) with high-osteogenic capacity. It is also reported that interference with MSC mobilization toward the periapex region in an AP mouse model led to enlargement of lesions, accompanied by decreased wound healing markers and increased inflammatory cytokines (Araujo-Pires et al., 2014). These findings indicate the involvement of MSCs in the repair and regeneration of oral inflammatory-related bone lesions. They also suggest that MSCs present promising targets for treating bone lesions, with great potential for modulating inflammation and promoting tissue regeneration. However, most studies have surveyed whole tissues to understand the transcriptomic and cellular profile of these diseases. Specific cell populations and their regulatory molecules, as well as the interaction among different cell populations, remain far from clear. The advances in single-cell technologies offer an unbiased approach for identifying heterogeneous cell subsets, tracking the trajectories of distinct cell clusters and uncovering regulatory relationships between genes (Hwang et al., 2018; Tang et al., 2009). In this study, we collected mandibular alveolar bone samples from control and AP in mice and subjected them to single-cell RNA sequencing (scRNA-seq). The atlas of the mandibular alveolar bone explored the distinct cell subsets and their expression profiles relevant to AP. We also investigated the relationship between MSCs and immune cell subsets. The results reveal the role of a subset of MSCs in inflammation, which showed increased frequency and which formed a self-supporting network. Moreover, MSCs exhibited upregulated osteogenic potential, which was confirmed in transgenic mouse models and human patients with chronic AP. These results advance our understanding of heterogeneity and interactions of alveolar bone cells in the pathogenesis of inflammatory-related bone diseases. Defining key cellular subsets such as MSCs and their biomarkers in inflamed tissue will be important for identifying new therapeutic targets for oral inflammatory-related bone diseases. Results Single-cell transcriptional profiling identified 15 discrete populations in homeostasis and chronic AP samples Individual cells were isolated from alveolar bone of healthy mice and mice with AP. We modeled AP using a well-established AP mouse model in which the mandibular first molar pulp was exposed and subsequently developed chronic AP over a 3 wk period (Taira et al., 2019). Bar-coded cDNA libraries from individual cells were obtained using the 10× Genomics Chromium Controller platform (Zheng et al., 2017; Figure 1A). The combined libraries from healthy and AP alveolar bone contained 15,148 individual cells. The median value of feature_RNA was between 1000 and 2000 (Figure 1—figure supplement 1B). After quality control filtering and removal of the batch effect between batches, the t-stochastic neighbor embedding (t-SNE) method was applied to reduce the dimensionality. Seurat's unbiased cluster detection algorithm identified 15 distinct cell populations (Figure 1B and C). Cluster-specific transcripts were utilized to annotate cell types with classic markers as described in a previous study (Lin et al., 2021). These included B cell (Cd79a), hematopoietic stem cell (HSC) (Cd34), MSC (Col1a1), natural killer (NK) cell (Klrd1), T cell (Cd3g), dendritic cell (Siglech), epithelial cell (Epcam), erythrocyte (Hbb-bt), macrophage (Adgre1), mast cell (Fcer1a), megakaryocyte (Gp1bb), monocyte (Ly6c2), myeloid progenitor (Mpo), neutrophil (S100a8), and pre-B cell (Vpreb1; Figure 1E and F). The top 20 enriched genes in each defined cluster were identified and compared (Figure 1G). Figure 1 with 1 supplement see all Download asset Open asset Identification of the single-cell atlas of alveolar bone using scRNA-sequencing (scRNA-seq) and unbiased clustering. (A) Schematic diagram of the experimental design. (B–C) t-Stochastic neighbor embedding (t-SNE) representation of aligned gene expression data in single cells extracted from mandibles of control mice (n=8340) and apical periodontitis (AP) mice (n=6808) showing 15 distinct clusters and cellular origin. (D) Relative abundance of 15 cell populations composing alveolar bone under healthy and AP conditions. (E) Expression of gene markers in distinct cell types. (F) Gene expression patterns projected onto t-SNE plots of marker genes. Scale: log-transformed gene expression. (G) Heatmap showing the 20 most upregulated genes (ordered by decreasing Padj value) in each cluster defined in B. Scale: log2 fold change. AP led to significant changes in frequency and transcriptional expression of cell populations All the identified cell clusters were present in both AP and control samples, but there were significant differences in the cellular compositions of particular clusters. T cell, B cell, NK cell, macrophage, epithelial cell, and MSC had significantly increased frequency in AP samples. Neutrophil, myeloid progenitor, monocyte, megakaryocyte, mast cell, HSC, and dendritic cell were markedly decreased (Figures 2A and 1D). Figure 2 Download asset Open asset Changes in frequency and transcriptional expression pattern in each cell population from control and apical periodontitis (AP) groups. (A) Bar plot of cells per cluster (AP versus control). Normalization to overall number of inputs per condition. Fisher's exact test with Bonferroni correction was used. *p<0.05, **p<0.01, and ****p<0.0001. All data were exhibited as mean ± SEM. (B) Violin plots of cluster-specific expression of representative genes. (C–G) Violin plots showing genes that significantly changed in each cluster from control and AP. AP is a complex inflammatory process involving innate and adaptive immune responses (Cotti et al., 2014). A variety of inflammatory cells such as neutrophils, mast cells, monocytes, macrophages, and lymphocytes are involved in periapical lesions, highlighting the direct involvement of the immune response in the pathogenesis of AP (Nair, 2004). Neutrophils are important components in the acute phase of AP as a first line of defense. But they are also important in the progression of AP by interacting with microorganisms, leading to tissue damage and chemotaxis (Braz-Silva et al., 2019). Single-cell differential expression analysis revealed that the most significantly enriched genes in neutrophils were various proinflammatory chemokines and cytokines. These included C-X-C motif chemokine ligand 2 (Cxcl2), C-C motif chemokine ligand 6 (Ccl6), NLR family pyrin domain containing 3 (Nlrp3), and Interleukin-1β (Il1b). Notably, we found that C-C motif chemokine receptor like 2 (Ccrl2) was upregulated in neutrophils during AP (Figure 2D). It is responsible for the innate defense against pathogens and is also involved in the regulation of neutrophil migration (Del Prete et al., 2017; Kolaczkowska and Kubes, 2013; Mantovani et al., 2011). Mast cells, monocytes, and macrophages have critical roles in the inflammatory infiltrate during chronic AP (Braz-Silva et al., 2019). The production of Interleukin-6 (Il6) was present in these cell populations with the highest expression level in mast cells. The pro-inflammatory cytokine IL-1β is a key mediator of host response to microbial infection and is associated with the persistence of AP (Morsani et al., 2011; Ng et al., 2008). We found Il1b transcripts in a series of cell types, such as monocyte, macrophage, mast cell, and neutrophil. Of these, macrophages had the highest Il1b expression. Another major cytokine, tumor necrosis factor (Tnf; Cotti et al., 2014), was detected in immunoresponsive cell clusters, such as monocyte, macrophage, mast cell, myeloid progenitor, neutrophil, and HSC, with the highest expression observed in the monocyte population (Figure 2B). Furthermore, gene signatures from monocytes showed that the interferon-induced transmembranes (IFITMs) protein 1 and 2 (Ifitm1 and Ifitm2; Figure 2E) were upregulated the most during AP. These factors have been associated with signal transduction of anti-inflammation activity in the immune system (Yánez et al., 2020). We also detected upregulated expression levels of Ccl9 in the monocyte population from AP. Ccl9 is an important cytokine and is involved in the survival of osteoclasts during the destruction of the periapical bone (Silva et al., 2007). Also, genes coding for pro-inflammatory calcium-binding S100 family proteins such as S100a9 and S100a11 had increased expression in the AP monocyte cluster (Figure 2E). Previous reports demonstrated that macrophages are capable of secreting pro- and anti-inflammatory substances which act on the development and repair of the AP lesions (Italiani and Boraschi, 2014; Shapouri-Moghaddam et al., 2018). Indeed, several genes encoding pro-inflammatory mediators, including Cxcl2, Cxcl16, Il1a, and Ptgs2, were upregulated in macrophages from AP samples (Figure 2C). Expression of anti-inflammatory-associated genes such as Ifitm1 and Ifitm2 was significantly increased in the AP macrophage cluster compared to control cells. Furthermore, Fcγ receptor IIB (Fcgr2b) was markedly upregulated in macrophages from AP samples. Fcgr2b is expressed in most tissue-resident macrophages (Gautier et al., 2012) and functions to inhibit Fcγ-dependent phagocytosis. It also inhibits release of cytokines such as IL-6, TNF-α, IL-1α, as well as neutrophil chemotactants (Clatworthy and Smith, 2004; Espéli et al., 2016). In addition, expression of Apolipoprotein E (Apoe), which can suppress the pro-inflammatory response (Jofre-Monseny et al., 2007), was significantly increased in the macrophage population (Figure 2C). These data indicated the activation of anti-inflammatory factors by macrophages during local inflammation by AP. The major classes of lymphocytes are T lymphocytes, B lymphocytes, and the NK cells. T and B lymphocytes comprise the majority of the inflammatory infiltrate in AP (Graunaite et al., 2012). A significant increase in the expression of inflammatory-associated genes, such as Srgn, Emb, Ctla4, and Il7r could be observed in the AP T cell population (Figure 2F). In the AP B lymphocytes cluster, inflammation-responsive genes (Fth1, Ftl1, Ebf1, and mt-Nd5) were upregulated (Figure 2G). Interestingly, Lars2, the gene encoding a mitochondrial leucyl tRNA synthase (Carminho-Rodrigues et al., 2020; 't Hart et al., 2005), was significantly upregulated in T and B lymphocytes, indicating changes in mitochondrial metabolism in both clusters. Inflammation induces osteoclasts differentiation leading to periapical alveolar bone destruction Bone destruction is a major pathological factor in chronic inflammatory diseases such as AP. Various cytokines including TNF-α, IL-1α, and IL-6 were released by immunocytes to recruit the osteoclast precursors and induce the maturation of osteoclasts (Lyu et al., 2022). We have detected osteoclast markers including Ctsk, Acp5, Mmp9, and Nfatc1 by scRNA-seq. Moreover, Csfr1, Cx3cr1, Itgam, and Tnfrs11a were used to identify osteoclast precursors. Markers of osteoclast and osteoclast precursors were highly expressed in the clusters of monocyte and macrophage (Figure 3A and B). Gene Ontology (GO) analysis showed that inflammation related immune reactions and bone resorption activity were significantly enriched in macrophage cluster (Figure 3C). To further study the differential trajectory of osteoclasts, pseudotime analysis was performed for the clusters of macrophage and monocyte. Two independent branch points were determined, and five monocyte/macrophage subclusters were scattered at different branches in the developmental tree (Figure 3D and G). The results showed that the monocyte cluster differentiated into the macrophage cluster (Figure 3E). During this trajectory, the gene expression pattern across pseudotime showed that osteoclastic genes, such as Ctsk, Acp5, Mmp9, Atp6v0d2, and Dcstamp, were progressively elevated (Figure 3F). Of note, we have observed a branch which was highly positive for Ctsk and Acp5 (Figure 3H), indicating the mature osteoclasts were differentiated from monocyte/macrophage lineage and contributed to inflammatory bone resorption during AP. We have also analyzed the expression of osteoclast related genes using the bulk RNA-seq library built on mandibular samples extracted from mice with AP. Markers of osteoclast and osteoclast precursors were significantly upregulated, confirming the osteoclasts activity in the inflammatory-related bone lesion (Figure 3I). Figure 3 with 1 supplement see all Download asset Open asset Inflammatory-related bone resorption under apical periodontitis (AP) situation. (A) The expression levels of markers of osteoclasts and osteoclast precursors. (B) Violin plots of the expression of osteoclastogenesis genes. (C) Gene Ontology (GO) enrichment analysis of the biological functions of macrophage cluster. (D) Trajectory order of the monocyte/macrophage populations by pseudotime value. (E) The differentiation trajectory of monocyte and macrophage clusters presented on a t-stochastic neighbor embedding (t-SNE) visualization. (F) The expression patterns of osteoclast markers during the trajectory of monocyte/macrophage populations. (G) Distribution of monocytes/macrophages on the developmental tree by clusters. (H) Heatmap of differential genes of three states. (I) Heatmap of genes associated with osteoclastogenesis in bulk RNA-seq analysis. AP leads to reduced transcriptionally inferred cellular interactions with an increased self-supporting network in MSCs We next sought to characterize the cell-cell communication related to the perturbation of signaling pathways detected in the AP samples by employing CellphoneDB (Efremova et al., 2020; Nagai et al., 2021). We identified a close interaction among MSC, macrophage, and dendritic cells under homeostasis conditions (Figure 4A). A similarly close communication was found among MSC, macrophage, and dendritic cells under inflammatory conditions (Figure 4B). Next, we compared the differential cell-cell interaction (CCI) network between AP and control samples using CrossTalkeR (Nagai et al., 2021). The results suggested that AP is associated with an overall decrease in cellular interactions. However, MSC intercellular communication with mast cells and monocytes is upregulated and accompanied by the highest number of interactions within MSCs themselves (Figure 4C). These results indicate that, although cell populations lose their normal physiological interactions, MSCs were able to establish a self-interacting network and coordinate with certain types of cells during chronic AP. Next, we ranked the individual ligands by the number of their interactions. Inflammatory-related proteins (Lgals9, Tnf, and Ccl4), extracellular matrix protein (Fn1), and protein involved in biomineralization (Spp1) were among the highest interactions. Also, Tgfb1, Vegfb, and Vegfa were in the top 10 most abundant ligands (Figure 4D). Bar plots were also generated to display the top 10 upregulated gene/cell pairs, showing that inflammation associated genes (Tnf/neutrophil, Ccl3/mast cell, Ccl3/monocyte, and Il1b/macrophage) and matrix related genes (Sele/MSC, Fn1/MSC, and Fn1/monocyte) were the most influential ligands during AP when compared to control state (Figure 4E). We used a Sankey plot to further focus on MSC-related interactions (Figure 4F). The results indicated that Sele was primarily directed by MSC toward the MSC cluster via multiple receptors including Glg1, Selplg, and Cd44. Moreover, Fn1 was secreted by MSC, monocyte, macrophage, and mast cells toward MSCs (Figure 4G). Of note, Sele and Fn1 are important cell adhesion molecules that mediate cell homing and migration (Frenette et al., 1998; To and Midwood, 2011). This was in accordance with the increased frequency of the MSC cluster (Figure 2A) as well as the upregulated cellular interaction among MSCs and other cell populations. Figure 4 Download asset Open asset Apical periodontitis (AP) suppressed transcriptionally inferred cellular interactions and increased a self-supporting network within the mesenchymal stem cell (MSC) cluster. (A–C) Network plot of ligand-receptor activity in control (A), AP (B), and AP versus control (C). (D) Bar plot of top 10 most abundant ligands in all inferred ligand-receptor interactions. (E) Ranking of ligand/source regarding communication gains in AP state. (F and G) Sankey plot listing all predicted source, receptor, and receiver interactions associated with Fn1 and Sele. scRNA-seq based identification of AP-associated MSC population MSCs represented the non-immune cell population in alveolar bone, constituting 1.76% of total identified cells. This cell population could be decomposed into four subclusters (Figure 5A). The most dominant subcluster was characterized by high expression of Prrx1, platelet-derived growth factor receptor β (Pdgfrb), and hematopoiesis supporting factors such as C-X-C motif chemokine 12 (Cxcl12) and angiopoietin (Angpt1). It was also characterized by osteogenic-related markers, such as Runt-related transcription factor 2 (Runx2), Sp7, and was thus classified as the MSC_osteolineage cells (OLCs). The other subclusters were identified as MSC_endothelial and Figure and B). The cell composition of the MSC cluster indicated an of subcluster during the subcluster was reduced (Figure Of note, we performed lineage tracing using and transgenic mouse models to identify stem cells in alveolar bone and the surrounding periodontal tissues et al., 2020; et al., et al., 2020; et al., 2020). The results suggested increased of and as well as upregulated and periodontal stem cells in AP lesions (Figure supplement 1A). We compared the MSC marker genes, identified among the four enrichment analysis confirmed that there are four MSC populations. were enriched for tissue system and osteoblast MSC_endothelial cells enrichment for and cells were enriched for of and suggested highly of cells, including regulation of protein from mitochondrial and (Figure supplement Figure with 3 see all Download asset Open asset Identification and of apical periodontitis mesenchymal stem cell (MSC) population and (A) t-Stochastic neighbor embedding (t-SNE) representation an of single cells within the MSC cluster. (B) Violin plots of MSC expression of representative genes. (C) The of four subclusters of MSC population were in control and AP groups. (D) Violin plots the changes in the expression of top upregulated genes in the MSC cluster. and of Sparc (E) and (F) in (G) of in periodontal stem cells (H) tracing analysis of and AP stimulates MSC differentiation toward osteoblast lineage cells We next compared the data from the MSC cluster between AP and control groups. the top upregulated genes, we observed a significant in the single cell expression levels of such as secreted protein that is and in and accompanied by a toward increased expression (Figure We their by and bulk RNA analysis. The results revealed that and were significantly upregulated, and to increase during AP (Figure supplement B). In confirmed the upregulated expression of osteogenic-related markers, such as Sparc and in the AP were increased of cells in the bone the protective of MSC to differentiate into and under AP conditions (Figure and F). tracing analysis further revealed that to AP lesions and differentiated into (Figure Moreover, using mouse we identified cells in the alveolar bone, confirming that inflammation could the progenitor cells in the bone and toward thus to the protective during AP (Figure We investigated the heterogeneity of MSCs by branch expression analysis and pseudotime analysis in 2 et al., The results revealed three distinct states. Of these, state 2 and state 3 represented differentiated cell populations Figure We the of the MSCs with to their states. We the pseudotime of each cluster cell in the state and the results indicate that with MSC_endothelial cells, MSCs were able to differentiate into and cells. cells may to an state (Figure Interestingly, in state as osteogenic exhibited the highest toward differentiation with upregulated of and (Figure Notably, this MSC population increased significantly in the AP (Figure A of the of the gene expression pattern across pseudotime revealed of osteogenic genes such as and during inflammation when compared to the state (Figure the MSC subcluster exhibited gene expression levels of osteogenic markers that increased during AP. These results were in accordance with our previous single-cell analysis of gene expression in the AP (Figure Figure 6 Download asset Open asset Apical periodontitis (AP) stimulates mesenchymal stem cell (MSC) differentiation toward osteoblast lineage cells. (A) analysis of the MSC (B) cell differentiation trajectory of MSC populations in control and AP groups. (C) Heatmap of differential genes of three states. (D) Bar plot of changes between AP and control in as identified in pseudotime analysis. Fisher's exact test with Bonferroni correction was used. ****p<0.0001. All data were exhibited as mean ± SEM. (E) between the pseudotime gene trajectories of MSC showed of and line and indicate marker levels were identified in alveolar bone from patients with AP We next osteogenesis within the MSC population was in human patients with AP. The of and were significantly enhanced in alveolar bone from patients with accompanied by in and (Figure Moreover, detected a of and in AP alveolar bone confirming higher osteogenic potential under AP conditions (Figure of markers in AP lesions from patients is with our previous an of osteogenic within the MSC subcluster in alveolar bone under AP conditions. Figure Download asset Open asset alveolar bone in apical periodontitis (AP) is associated with higher subcluster (A) Gene expression of and in samples of healthy and AP patients showed an upregulated expression in AP in healthy and in AP All data are as the mean ± SEM. (B–C) of and in bone of human alveolar bone exhibited high osteogenic protein levels in AP. 10 In summary, the current study the of MSCs and immunoresponsive cells under healthy and chronic AP including heterogeneity in their of We that this analysis a

ObjectiveOsteoarthritis (OA) presents cartilage damage in addition to chronic inflammation. However, self-recovery of damaged cartilage in an inflammatory environment is not possible. Mesenchymal stem cells (MSCs) in the bone marrow are a source of regenerative repair of damaged cartilage. To date, whether intra-luminal administration of the bone marrow can delay the progression of OA is still unknown. This study, therefore, aimed to explore the role of intra-bone marrow injection of Magnesium isoglycyrrhizinate (MgIG) in delaying the OA progression and to investigate the underlying mechanism.MethodsRabbit OA models were established using the anterior cruciate ligament transection method while a catheter was implanted into the bone marrow cavity. 1 week after surgery, MgIG treatment was started once a week for 4 weeks. The cartilage degradation was analyzed using hematoxylin–eosin staining, Masson’s trichrome staining and Alcian blue staining. Additionally, the pro-inflammatory factors and cartilage regeneration genes involved in the cartilage degeneration and the underlying mechanisms in OA were detected using enzyme-linked immunosorbent assay, quantitative real-time PCR (qRT-PCR) and Western blotting.ResultsThe results of histological staining revealed that intra-bone marrow injection of MgIG reduced degeneration and erosion of articular cartilage, substantially reducing the Osteoarthritis Research Society International scores. Furthermore, the productions of inflammatory cytokines in the bone marrow cavity and articular cavity such as interleukin-1β(IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were inhibited upon the treatment of MgIG. At the same time, the expression of alkaline phosphate, tartrate-resistant acid phosphatase-5b (TRAP-5b) and C-telopeptides of type II collagen (CTX-II) in the blood also decreased and was positively correlated. On the contrary, cartilage-related genes in the bone marrow cavity such as type II collagen (Col II), Aggrecan (AGN), and SRY-box 9 (SOX9) were up-regulated, while matrix metalloproteinase-3 (MMP-3) was down-regulated. Mechanistically, MgIG was found to exert an anti-inflammatory effect and impart protection to the cartilage by inhibiting the NF-κB pathway.ConclusionIntra-bone marrow injection of MgIG might inhibit the activation of the NF-κB pathway in the progression of OA to exert an anti-inflammatory effect in the bone marrow cavity and articular cavity, thereby promoting cartilage regeneration of MSCs in the bone marrow, making it a potential new therapeutic intervention for the treatment of OA.

Mesenchymal stem cell (MSC) transplantation has emerged as a promising approach for bone regeneration. Importantly, the beneficial effects of MSCs can be improved by modulating the expression levels of specific genes to stimulate MSC osteogenic differentiation. We have previously shown that Smurf1 silencing by using Locked Nucleic Acid-Antisense Oligonucleotides, in combination with a scaffold that sustainably releases low doses of BMP-2, was able to increase the osteogenic potential of MSCs in the presence of BMP-2 doses significantly smaller than those currently used in the clinic. This would potentially allow an important reduction in this protein in MSs-based treatments, and thus of the side effects linked to its administration. We have further improved this system by specifically targeting the Wnt pathway modulator Sfrp1. This approach not only increases MSC bone regeneration efficiency, but is also able to induce osteogenic differentiation in osteoporotic human MSCs, bypassing the need for BMP-2 induction, underscoring the regenerative potential of this system. Achieving successful osteogenesis with the sole use of LNA-ASOs, without the need of administering pro-osteogenic factors such as BMP-2, would not only reduce the cost of treatments, but would also open the possibility of targeting these LNA-ASOs specifically to MSCs in the bone marrow, allowing us to treat systemic bone loss such as that associated with osteoporosis.