RESUMO
Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.
Assuntos
Calcificação Fisiológica , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ligamento Periodontal/citologia , Calcificação Fisiológica/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Osteogênese/genética , Ftalimidas/farmacologia , Triptofano/análogos & derivados , Triptofano/farmacologia , Regulação para Cima/efeitos dos fármacos , Adulto JovemRESUMO
Mesenchymal stem cells are candidates for therapeutic strategies in periodontal repair due to their osteogenic potential. In this study, we identified epigenetic markers during osteogenic differentiation, taking advantage of the individual pattern of mesenchymal cells of the periodontal ligament with high (h-PDLCs) and low (l-PDLCs) osteogenic capacity. We found that the involvement of non-coding RNAs in the regulation of the RUNX2 gene is strongly associated with high osteogenic potential. Moreover, we evaluated miRs and genes that encode enzymes to process miRs and their biogenesis. Our data show the high expression of the XPO5 gene, and miRs 7 and 22 observed in the l-PDLCs might be involved in acquiring osteogenic potential, suppressing RUNX2 gene expression. Further, an inversely proportional correlation between lncRNAs (HOTAIR and HOTTIP) and RUNX2 gene expression was observed in both l- and h-PDLCs, and it was also related to the distinct osteogenic phenotypes. Thus, our results indicate the low expression of XPO5 in h-PDLC might be the limiting point for blocking the miRs biogenesis, allowing the high gene expression of RUNX2. In accordance, the low expression of miRs, HOTAIR, and HOTTIP could be a prerequisite for increased osteogenic potential in h-PDLCs. These results will help us to better understand the underlying mechanisms of osteogenesis, considering the heterogeneity in the osteogenic potential of PDLCs that might be related to a distinct transcriptional profile of lncRNAs and the biogenesis machinery.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/metabolismo , Osteogênese , Ligamento Periodontal/citologia , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Carioferinas/genética , Carioferinas/metabolismo , MicroRNAs/genética , Ligamento Periodontal/metabolismo , Fenótipo , RNA Longo não Codificante/genética , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Transcrição Gênica , Transcriptoma , Adulto JovemRESUMO
A more accurate understanding of the molecular mechanisms and signaling pathways underpinning human mesenchymal stem cell (MSC) plasticity and differentiation properties is pivotal for accomplishing solid and diligent translation of MSC-based experimental therapeutics and clinical trials to broad clinical practice. In addition, this knowledge enables selection of MSC subpopulations with increased differentiation potential and/or use of exogenous factors to boost this potential. Here, we report that CD105 (ENG) is a predictive biomarker of osteogenic potential in two types of MSCs: stem cells from human exfoliated deciduous teeth (SHED) and human adipose-derived stem cells (hASC). We also validate that CD105 can be used to select and enrich for subpopulations of SHED and hASC with higher in vitro osteogenic potential. In addition, we show that hsa-mir-1287 regulates CD105 expression, and propose that fine-tuning hsa-mir-1287 levels could be used to control osteopotential in SHED. These findings provide better discernment of the molecular bases behind MSC osteogenic plasticity and open up new perspectives to leverage osteogenic potential in MSCs by modulation of a specific miRNA.
Assuntos
Endoglina/metabolismo , MicroRNAs/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Criança , Endoglina/genética , Citometria de Fluxo , Humanos , Imunofenotipagem , Fator de Crescimento Insulin-Like II/farmacologia , MicroRNAs/genética , Osteogênese/genética , Osteogênese/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa Gomes, commonly known as Mangabeira, is a Brazilian native fruit tree belonging to the Apocynaceae family. In folk medicine, the latex obtained from Mangabeira's trunk has been used as an adjunct therapy for bone fractures. Few pharmacological studies on the Hancornia speciosa latex have been developed and despite its popular use for bone healing there is no data about its biological effect on bone. AIM OF THE STUDY: The present study aimed to investigate the osteogenic potential of Hancornia speciosa latex in rat calvaria, as well as its phytochemical profile. MATERIALS AND METHODS: A neutral gel composition containing 5% latex was topical applied to a critical size bone defect and over intact calvaria of rats. Areas of newly formed bone on the borders of the defect and of calvaria periosteum were quantified, as well as the percentage of BrdU-positive cells and total cells in the periosteum at different periods of time after latex application. The cytotoxicity of the latex aqueous phase was evaluated in rat calvarial cells in vitro by MTT assay and its phytochemical profile was investigated by ESI-MS/MS. RESULTS: The area of newly formed bone on the borders of the calvaria defect was larger in rats that received latex at 15 and 30 days of healing. After 3 days of latex application over the intact calvaria, the periosteum area was increased and newly formed bone was observed after 5 and 11 days. There was also an increase in periosteum cell proliferation and population followed latex application on calvaria (p<0.05). The latex aqueous phase limited rat calvarial cell viability in vitro in concentrations larger than 0.6mg/mL. Chlorogenic acid and naringenin-7-O-glucoside were identified in the latex aqueous phase, along with catechin and procyanidin compounds. CONCLUSION: There was a stimulus for periosteum cell proliferation and bone formation when Hancornia speciosa latex was topically applied on rat calvaria. In addition, chlorogenic acid and naringenin-7-O-glucoside present in Hancornia speciosa latex may contribute to its effects on bone formation.