Transcriptome profile of highly osteoblastic/cementoblastic periodontal ligament cell clones

Abstract Heterogeneous cell populations of osteo/cementoblastic (O/C) or fibroblastic phenotypes constitute the periodontal dental ligament (PDL). A better understanding of these PDL cell subpopulations is essential to propose regenerative approaches based on a sound biological rationale. Objective Our study aimed to clarify the differential transcriptome profile of PDL cells poised to differentiate into the O/C cell lineage. Methodology To characterize periodontal-derived cells with distinct differentiation capacities, single-cell-derived clones were isolated from adult human PDL progenitor cells and their potential to differentiate into osteo/cementoblastic (O/C) phenotype (C-O clones) or fibroblastic phenotype (C-F clones) was assessed in vitro. The transcriptome profile of the clonal cell lines in standard medium cultivation was evaluated using next-generation sequencing technology (RNA-seq). Over 230 differentially expressed genes (DEG) were identified, in which C-O clones showed a higher number of upregulated genes (193) and 42 downregulated genes. Results The upregulated genes were associated with the Cadherin and Wnt signaling pathways as well as annotated biological processes, including "anatomical structure development" and "cell adhesion." Both transcriptome and RT-qPCR showed up-regulation of WNT2, WNT16, and WIF1 in C-O clones. Conclusions This comprehensive transcriptomic assessment of human PDL progenitor cells revealed that expression of transcripts related to the biological process "anatomical structure development," Cadherin signaling, and Wnt signaling can identify PDL cells with a higher potential to commit to the O/C phenotype. A better understanding of these pathways and their function in O/C differentiation will help to improve protocols for periodontal regenerative therapies.

Isolation and characterization of a human cementocyte-like cell line, HCY-23

Abstract Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.

Localización de las proteínas específicas del cemento radicular CEMP1 y CAP en células neoplásicas / Immunolocalization of cementum specific proteins CEMP1 and CAP in tumor cells

Introducción: Las proteínas CEMP1 y CAP presentes en los cementoblastos y sus progenitores contribuyen a los procesos de mineralización en tejidos del ligamento periodontal, incluyendo la migración y la proliferación de fibroblastos gingivales; sin embargo su papel y relación con procesos neoplásicos no se han estudiado a profundidad. Para lograr un mejor entendimiento de la posible contribución de estas proteínas en los procesos tumorales, particularmente en las metástasis óseas, se investigó su expresión y localización en tejidos y líneas celulares de cáncer humano. Materiales y métodos: Trece casos de cáncer de próstata y mama que desarrollaron enfermedad metastásica ósea fueron analizados por medio de inmunohistoquímica; mientras que la expresión de las proteínas en dos líneas celulares de carcinoma de próstata (PC-3) y mama (MCF-7) se estudió por medio de ensayos de Western Blot. Resultados: Los tejidos de cáncer revelaron expresión citoplasmática y ocasionalmente nuclear de CAP en células tumorales y estructuras glandulares pequeñas, así como en el citoplasma de los fibroblastos estromales adyacentes al frente de invasión tumoral. En lo correspondiente a CEMP1, su expresión se localizó en el citoplasma de las células tumorales de 5 casos, pero no en el estroma. Ensayos de Wester Blot mostraron expresión de CEMP1 en las células PC-3 y MCF-7; y de CAP en las MCF-7. Conclusiones: Los resultados muestran que las proteínas de cemento radicular CEMP1 y CAP se expresan en tejidos neoplásicos y células neoplásicas, y que posiblemente contribuyen en ciertas condiciones patológicas como el cáncer metastásico en humanos.

Introduction: CEMP1 and CAP are recognized as cementum proteins, they appear to be limited to cementoblasts and their progenitors, and participate in the mineralization process of periodontal ligament tissues, including the proliferation and migration of periodontal ligament fibroblasts. However, their contribution in neoplastic processes had not been explored. In the present study, we investigated their protein expression and localization in cancer tissues and cells. Materials and Methods: CEMP1 and CAP expressions were analyzed immunohistochemically in 13 cancer cases with bone metastasis. In addition, Wester Blot essays were use to detect expression of the proteins in the prostate (PC-3) and mama (MCF-7) cancer cell lines. Results: CAP expression was detected in all tissues examined. Strong cytoplasmatic and rarely nuclear staining was found in small tumor nests, glandular structures and, in the stromal fibroblasts at the immediate vicinity of the tumor nests. CEMP1 was found in the cytoplasm of tumor cells in 5 cases, but its expression was negative in the stromal tissues. Also, cancer lines PC-3 and MCF-7 showed CEMP1 expression; however, CAP expression was observed only in MCF-7 cells. Conclusions: The results suggest that CEMP1 and CAP are present in tissues other that cementum and possibly contribute to pathological conditions such as metastatic cancer.

Cyclosporin a-induced new cementum formation: a morphometric evaluation in the periapical region of rats

Cyclosporin A (CsA) is a potent immunosuppressor used in organ transplantation and in the management of various autoimmune diseases. Recent studies have shown that CsA stimulates deposition of cementum on root surfaces. The aim of this study was to evaluate the periapical cementum thickness and the apical foramen width in CsA-treated rats. Rats weighing 50 g were treated with a daily injection of 10 mg/kg body weight of CsA in the chow for 60 days. The cementum of the mandibular 1st molars was histologically and morphometricaly examined by analysis of 5-æm-thick serial buccolingual paraffin sections stained with hematoxylin and eosin. Histometric and stereologic analyses revealed the presence of large amounts of cementum in all root surfaces, particularly abundant in the periapical region and obliterating the foramen. The volume density of cementoblasts did not increase. Five to 90 days after the termination of CsA therapy, there was no reduction of cementum thickness. These results suggest that cementum deposition is not reversible after cessation of CsA treatment.

Ciclosporina A (CsA) é um potente imunossupressor usado no transplante de órgãos e no tratamento de várias doenças auto-imunes. Recentes estudos têm demonstrado que a CsA estimula a deposição de cemento na superfície radicular. O objetivo deste estudo foi de avaliar a espessura do cemento periapical e largura do forame apical em ratos tratados com CsA. Os ratos pesavam 50 g e foram tratados com doses diárias de 10 mg/kg de peso corporal de CsA no período de 60 dias. O cemento do primeiro molar inferior foi examinado histologicamente e morfometricamente por análises de cortes em parafina com 5æm de espessura no sentido vestíbulo-lingual e corados com hematoxilina e eosina. As análises histométricas e estereológicas revelaram a presença de largos depósitos de cemento em todas as superfícies radiculares, particularmente maior na região periapical e obliterando o forame. A densidade volumétrica dos cementoblastos não foi aumentada. No período de 5 a 90 dias após o término da terapia com CsA, não houve redução na espessura do cemento. Estes resultados sugerem que o depósito de cemento não é reversível após o tratamento com CsA ser cessado.