Inflammatory environment induces gingival tissue-specific mesenchymal stem cells to differentiate towards a pro-fibrotic phenotype.

BACKGROUND INFORMATION: Human gingival tissues are prone to hyperplasia under inflammatory stimuli. We have identified gingival tissue-specific mesenchymal stem cells (GMSCs) and found their functional change being correlated with drug-induced gingival hyperplasia. However, whether these cells exhibit characteristics of pro-fibrotic phenotype under inflammatory condition remains unknown. RESULTS: GMSCs isolated from human normal gingival tissues (N-GMSC) and inflammatory gingival tissues (I-GMSC) were cultured in vitro, representative cytokines were added to simulate the in vivo inflammatory environment. Under the influence of the inflammatory cytokines, GMSCs exhibited higher rate of proliferation than those under normal condition, while their potential for osteogenic and adipogenic differentiation was suppressed. The expression of matrix metalloproteinases (MMP)-1, MMP-2, IL-1, IL-6, TNF-α and type 1 collagen was significantly higher in I-GMSCs than in N-GMSCs. Furthermore, compared with dental pulp stem cells, GMSCs showed different pattern of gene expression and extracellular matrix formation in inflammatory environment. CONCLUSIONS: Inflammatory microenvironment induces GMSCs to differentiate towards a pro-fibrotic phenotype, which could underlie the hyperplastic appearance of inflammatory gingiva.

The effect of the cleidocranial dysplasia-related novel 1116_1119insC mutation in the RUNX2 gene on the biological function of mesenchymal cells.

Bone extracellular matrix deposition or bone formation by differentiated osteoblasts begins at late stage during bone formation and lasts throughout life. Human mesenchymal stem cells (MSCs) from bone marrow or dental pulp can respectively differentiate into osteoblasts and odontoblasts in vitro. However, the relationship between MSCs and bone/tooth development in cleidocranial dysplasia (CCD) patient is still unclear. In this study, we investigated a patient with CCD, which is an autosomal-dominant, heritable skeletal disease caused by runt-related transcription factor 2 gene (RUNX2) mutation and is characterized by bone and dental anomalies. We found that the mutation is localized at c. 1116_1119insC, p. Q374fsX384 and the proliferative ability and osteogenic potential of the MSCs isolated from the bone marrow and dental pulp of the patient (RUNX2(+/m)) were decreased compared to normal individuals (RUNX2(+/+)). Furthermore, we were unable to recover the differentiation potential of RUNX2(+/m) MSCs isolated from the bone marrow (BMMSCs) upon manipulation of the Wnt/ß-catenin pathway, which plays a critical role in stem/progenitor cell self-renewal and adult human MSCs differentiation. In conclusion, we identified a novel insertion/frameshift mutation in the RUNX2 gene that caused a typical CCD phenotype and altered the biological function of RUNX2(+/m) MSCs. The reduced ability of MSCs to differentiate into osteoblasts might provide an explanation for the defects of bone and teeth in the CCD patient. Finally, we demonstrated that manipulation of the Wnt/ß-catenin signaling pathway could not overcome this absence.

Heterogeneous dental follicle cells and the regeneration of complex periodontal tissues.

Dental follicle cells (DFCs) are a heterogeneous population that exhibit a variety of phenotypes. However, it remains unclear whether DFCs can maintain stem cell characteristics, or mediate tissue-regeneration to form single or complex tissues in the periodontium, after long-term culturing. Therefore, DFCs were isolated from human impacted molars (HIM-DFCs), passaged >30 times, and then evaluated for their heterogeneity and multipotential differentiation. Morphology, proliferation, epitope profile, and mineralization characteristics of clones derived from single HIM-DFCs in vitro were also assayed. HIM-DFCs (passage #30) were found to be positive for the heterogeneous markers, Notch-1, stro-1, alkaline phosphomonoesterase (ALP), type I collagen (COL-I), type III collagen (COL-III), and osteocalcine. Moreover, passage #30 of the HDF1, 2, and 3 subclone classes identified in this study were found to express high levels of the mesenchymal stem cells markers, CD146 and Stro1. HDF3 subclones were also associated with the strongest ALP staining detected, and strongly expressed osteoblast and cementoblast markers, including COL-I, COL-III, bone sialoprotein (BSP), and Runx2. In contrast, HDF1 subclone analyzed strongly expressed COL-I and COL-III, yet weakly expressed BSP and Runx2. The HDF2 subclone was associated with the strongest proliferative capacity. To evaluate differentiation characteristics in vivo, these various cell populations were combined with ceramic bovine bone and implanted into subcutaneous pockets of nude mice. The 30th passage of subclone HDF1 and 3 were observed to contribute to fiber collagens and the mineralized matrix present, respectively, whereas HDF2 subclones were found to have a minimal role in these formations. The formation of a cementum-periodontal ligament (PDL) complex was observed 6 weeks after HIM-DFCs (passage #30) were implanted in vivo, thus suggesting that these cells maintain stem cell characteristics. Therefore, subclone HDF1-3 may be related to the differentiation of fibroblasts in the PDL, undifferentiated cells, and osteoblasts and cementoblasts, respectively. Overall, this study is the first to amplify HIM-DFCs and associated subclones with the goal of reconstructing complex or single periodontium. Moreover, our results demonstrate the potential for this treatment approach to address periodontal defects that result from periodontitis, or for the regeneration of teeth.

Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root.

Tissue engineering strategies to reconstruct tooth roots are an effective therapy for the treatment of tooth loss. However, strategies to successfully regenerate tooth roots have not been developed and optimized. In the present study, rat dental follicle stem cells (DFCs) were characterized, followed by a thorough investigation of tooth roots regeneration for a combination of DFCs seeding cells, treated dentin matrix (TDM) scaffolds, and an inductive alveolar fossa microenvironment. Eighteen clones derived from single DFCs were harvested; however, only three clones were amplified successfully more than five passages and 90-95 days in culture. Following 270 days or 30 passages, the heterogeneous DFCs showed suitable characteristics for seeding cells to regenerate tooth roots. However, various features, such as variable proliferation rates, differentiation characteristics, apoptosis rates, and total lifespan were observed in DFCs and the three clones. Importantly, upon transplantation of DFCs combined with TDM for four weeks, root-like tissues stained positive for markers of dental pulp and periodontal tissues were regenerated in the alveolar fossa, but not in the skull and omental pockets. These results indicate that tooth roots were successfully regenerated and suggest that the combination of DFCs with TDM in the alveolar fossa is a feasible strategy for tooth roots regeneration. This strategy could be a promising approach for the treatment of clinical tooth loss and provides a perspective with potential applications to regeneration of other tissues and organs.

MiR-17 modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis.

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis, are the most common causes of bone tissue destruction. Recently, human periodontal ligament tissue-derived mesenchymal stem cells (PDLSCs), a population of multipotent stem cells, have been used to reconstruct tissues destroyed by chronic inflammation. However, the impact of the local inflammatory microenvironment on tissue-specific stem cells and the mechanisms controlling the effects of the local inflammatory environment remain poorly understood. In this study, we found that the multidifferentiation potential of mesenchymal stem cells (MSCs) isolated from periodontitis-affected periodontal ligament tissue (P-PDLSCs) was significantly lower than that of MSCs isolated from healthy human periodontal ligament tissue (H-PDLSCs). Inflammation in the microenvironment resulted in an inhibition of miR-17 levels, and a perturbation in the expression of miR-17 partly reversed the differentiation potential of PDLSCs in this microenvironment. Furthermore, inflammation in the microenvironment promoted the expression of Smad ubiquitin regulatory factor one (Smurf1), an important negative regulator of MSC osteogenic differentiation. Western blotting and 3' untranslated regions (3'-UTR) reporter assays confirmed that Smurf1 is a direct target of miR-17 in PDLSCs. Our data demonstrate that excessive inflammatory cytokine levels, miR-17, and Smurf1 were all involved in a coherent feed-forward loop. In this circuit, inflammatory cytokines led to direct activation of Smurf1 and downregulation of miR-17, thereby increasing degradation of Smurf1-mediated osteoblast-specific factors. The elucidation of the molecular mechanisms governing MSC osteogenic differentiation in a chronic inflammatory microenvironment could provide us with a better knowledge of chronic inflammatory disorder and improve stem cell-mediated inflammatory bone disease therapy.

High levels of ß-catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway.

Periodontal ligament stem cells (PDLSCs), a new population of mesenchymal stem cells (MSCs), have been isolated from the periodontal ligament (PDL). The capacity of multipotency and self-renewal makes them an excellent cell source for bone regeneration and repair. However, their bone-regeneration ability could be awakened in inflammatory microenvironments, which may be the result of changes in their differentiation potential. Recently, genetic evidences has shown that the Wnt pathway plays an important role in bone homeostasis. In this study we have determined the specific role of ß-catenin in osteogenic differentiation of PDLSCs obtained from inflammatory microenvironments (P-PDLSCs). The inflammatory microenvironment, while inhibiting osteogenic differentiation potential, promotes proliferation of MSCs. A higher the level of ß-catenin in P-PDLSCs than in H-PDLSCs (PDLSCs obtained from a healthy microenvironment) resulted in the same disparity in canonical Wnt signaling pathway activation between each cell type. Here we show that activation of ß-catenin suppresses the noncanonical Wnt/Ca(2+) pathway, leading to increased proliferation but reduced osteogenic differentiation of P-PDLSCs. Downregulation of the levels of ß-catenin by treatment with dickkopf-1 (DKK-1) leads to activation of the noncanonical Wnt/Ca(2+) pathway, which, in turn, results in the promotion of osteogenic differentiation in P-PDLSCs. Interestingly, ß-catenin can affect both the canonical Wnt/ß-catenin pathway and the noncanonical Wnt/Ca(2+) pathway. Our data indicate that ß-catenin plays a central role in regulating osteogenic differentiation of MSCs in inflammatory microenvironments. Given the important role of Wnt signaling in osteogenic differentiation, it is possible that agents that can modify this pathway may be of value in bone regeneration by MSCs in chronic inflammatory microenvironments.