Spontaneous destructive periodontitis and skeletal bone damage in transgenic mice carrying a human shared epitope-coding HLA-DRB1 allele.

OBJECTIVE: Shared epitope (SE)-coding DRB1 alleles are associated with bone erosion in several diseases, including rheumatoid arthritis (RA) and periodontal disease (PD), but the underlying mechanism is unknown. We have recently identified the SE as an osteoclast-activating ligand. To better understand the biological effects of the SE in vivo, here we sought to determine whether it can facilitate spontaneous bone damage in naïve mice. METHODS: 3-month old naïve transgenic mice that carry the human SE-coding allele DRB1*04:01, or a SE-negative allele DRB1*04:02 were studied. Bone tissues were analysed by micro-CT, and the tooth-supporting tissues were studied by histology, immunohistochemistry and immunofluorescence. Serum biomarkers were determined by ELISA. RESULTS: Transgenic mice expressing the SE-coding DRB1*04:01 allele, but not mice carrying the SE-negative allele DRB1*04:02, showed spontaneous PD associated with interleukin (IL)-17 overabundance and periostin disruption. Mandibular bone volumetric and mineralisation parameters were significantly lower in SE-positive mice, and alveolar bone resorption was significantly increased in these mice. SE-positive mice also had more slender tibiae, and their marrow, cortical and total areas were lower than those of SE-negative mice. Additionally, significantly increased serum IL-17, tumour necrosis factor-α and osteoprotegrin levels were found in SE-positive mice, while their receptor activator of nuclear factor κ-B ligand levels were significantly lower. CONCLUSIONS: A human SE-coding allele increases the propensity to spontaneous bone-destructive periodontal inflammation and skeletal bone damage in transgenic mice. These findings provide new insights into the previously documented but poorly understood association of the SE with accelerated bone erosion in RA and several other human diseases.

Preliminary insight into the periostin leverage during periodontal tissue healing.

BACKGROUND: Tissue repair and regeneration is assisted by the efficient coordination of cell and extracellular matrix interactions mediated by matricellular molecules such as periostin. Given its high expression around the teeth, the periodontal organ represents an ideal system to capture the protein dynamics during wound healing. METHODS: An observational prospective case-control study was designed to characterize periostin changes over time after periodontal surgery in tissue, oral fluids and serum by histological, protein and mRNA analyses. RESULTS: Histological analysis showed lower periostin with a diffuse local distribution pattern in disease patients. Levels of periostin in gingival crevicular fluid (GCF) increased over time for both groups, more noticeably in the periodontitis subjects. A transient and subtle change in circulating periostin levels was also noticed. The mRNA periostin levels contrasted with the protein levels and may indicate the underlying post-transcriptional regulatory process during chronic inflammation. Levels of known periodontal disease biomarkers such as IL-ß, IL1-α, TNF-α, MIP-1α and CRP served as tissue stability markers and complemented the clinical parameters recorded. CONCLUSION: The transient local increase in GCF periostin after eliminating the local etiology in periodontally affected sites suggests its importance in the maturation and stability of the connective tissue. The decreasing levels observed as the tissue healed highlight its spatial/temporal significance.

Standardized in vivo model for studying novel regenerative approaches for multitissue bone-ligament interfaces.

The regeneration of the original structure and function of bone-ligament interfaces remains a major challenge in biomedical research. A preclinical model that maintains physiologic mechanical loads and controls for other external factors, such as microbial influence, is of great value for testing novel regenerative materials, provided that studies are performed by highly trained researchers with proper regard for animal welfare. The tooth root fenestration preclinical model is an ideal tool for hard tissue evaluation by micro-computed tomography, histological techniques and RNA analyses. The procedure starts with an extraoral incision lateral to the mandible and reflection of the masseter muscle. Superficial lateral mandibular bone is removed with standardized dimensions to expose the roots of the teeth and to eliminate periodontal ligament and cementum to expose the tooth dentin. The testing material can subsequently be applied to the defect and the flap can be repositioned and secured back in place. At specific time points, samples are collected and processed according to the subsequent analyses to be performed, which can include descriptive histology, histomorphometry, immunostaining, 3D bone imaging, electron microscopy, gene expression analyses and safety assessments.

The expression of periostin in dental pulp cells.

BACKGROUND AND OBJECTIVE: Dental pulp repair is a common process triggered by microbial and mechanical challenges. Matricellular modulators, such as periostin, are key for extracellular matrix stability and tissue healing. In the scope of the dental pulp, periostin expression has been reported during development and active dentinogenesis. However, the specific dental pulp cell population capable of expressing periostin in response to known regulators has not been clearly defined. Among the different relevant cell populations (i.e., stem cells, fibroblasts and pre-odontoblasts) potentially responsible for periostin expression in the dental pulp, this study aimed to determine which is the primary responder to periostin regulators. METHODS: Human dental pulp stem cells (DPSCs), human dental pulp fibroblasts (DPFs), and rat odontoblast-like cells (MDPC-23) were treated with different concentrations of TGF-ß1 or different regimens of biomechanical stimulation to evaluate periostin expression by qRT-PCR, Western blot and ELISA. Statistical analyses were performed by Student's t-test and ANOVA with Fisher's LSD post hoc tests (p ≤ 0.05). RESULTS: DPSC and MDPC-23 showed a statistically significant increase in periostin mRNA expression after exposure to TGF-ß1 for 48 h. TGF-ß1 also up-regulated periostin protein levels in DPSC. However, periostin significantly down-regulated protein expression in DPF. Different regimens of biomechanical stimulation showed different patterns in protein and mRNA periostin expression. CONCLUSIONS: Expression of periostin was identified in each of the analysed dental pulp cell lines, which can be regulated by TGF-ß1 and biomechanical stimulation. Overall, DPSCs are the most responsive cells to stimulation.

Periostin responds to mechanical stress and tension by activating the MTOR signaling pathway.

Current knowledge about Periostin biology has expanded from its recognized functions in embryogenesis and bone metabolism to its roles in tissue repair and remodeling and its clinical implications in cancer. Emerging evidence suggests that Periostin plays a critical role in the mechanism of wound healing; however, the paracrine effect of Periostin in epithelial cell biology is still poorly understood. We found that epithelial cells are capable of producing endogenous Periostin that, unlike mesenchymal cell, cannot be secreted. Epithelial cells responded to Periostin paracrine stimuli by enhancing cellular migration and proliferation and by activating the mTOR signaling pathway. Interestingly, biomechanical stimulation of epithelial cells, which simulates tension forces that occur during initial steps of tissue healing, induced Periostin production and mTOR activation. The molecular association of Periostin and mTOR signaling was further dissected by administering rapamycin, a selective pharmacological inhibitor of mTOR, and by disruption of Raptor and Rictor scaffold proteins implicated in the regulation of mTORC1 and mTORC2 complex assembly. Both strategies resulted in ablation of Periostin-induced mitogenic and migratory activity. These results indicate that Periostin-induced epithelial migration and proliferation requires mTOR signaling. Collectively, our findings identify Periostin as a mechanical stress responsive molecule that is primarily secreted by fibroblasts during wound healing and expressed endogenously in epithelial cells resulting in the control of cellular physiology through a mechanism mediated by the mTOR signaling cascade.

Tumor necrosis factor-α and Porphyromonas gingivalis lipopolysaccharides decrease periostin in human periodontal ligament fibroblasts.

BACKGROUND: Periostin is a matricellular protein essential for tissue integrity and maturation and is believed to have a key function as a modulator of periodontal ligament (PDL) homeostasis. The aim of this study is to evaluate whether periodontal disease-associated pathogen-related virulence factors (endotoxins/lipopolysaccharides [LPS]) and proinflammatory cytokines alter the expression of periostin in PDL cells. METHODS: Human PDL cultures were exposed to inflammatory mediators (tumor necrosis factor-α [TNF-α]), bacterial virulence factors (Porphyromonas gingivalis LPS) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. Periostin and TGF-ß inducible gene clone H3 (ßIGH3) mRNA expression from cell lysates were analyzed. Periostin and ßIGH3 proteins were also detected and semiquantified in both cell lysates and cell culture supernatants by Western blot. In addition, periostin localization by immunofluorescence was performed. Analysis of variance and Fisher tests were used to define the statistical differences among groups (P <0.05). RESULTS: In a mechanically challenged environment, periostin protein was more efficiently incorporated into the matrix compared to the non-loaded controls (higher levels of periostin in the supernatant in the non-loaded group). Interestingly, chronic exposure to proinflammatory cytokines and/or microbial virulence factors significantly decreased periostin protein levels in the loaded cultures. There was greater variability on ßIGH3 levels, and no particular pattern was clearly evident. CONCLUSIONS: Inflammatory mediators (TNF-α) and bacterial virulence factors (P. gingivalis LPS) decrease periostin expression in human PDL fibroblasts. These results support a potential mechanism by which periostin alterations could act as a contributing factor during periodontal disease progression.

Gene expression dynamics during bone healing and osseointegration.

BACKGROUND: Understanding the molecular features of bone repair and osseointegration may aid in the development of therapeutics to improve implant outcomes. The purpose of this investigation is to determine the gene expression dynamics during alveolar bone repair and implant osseointegration. METHODS: An implant osseointegration preclinical animal model was used whereby maxillary defects were created at the time of oral implant placement, while a tooth extraction socket healing model was established on the contralateral side of each animal. The surrounding tissues in the zone of the healing defects were harvested during regeneration for temporal evaluation using histology, immunohistochemistry, laser capture microdissection, and quantitative reverse transcription-polymerase chain reaction for the identification of a panel of 17 putative genes associated with wound repair. RESULTS: In both models, three distinct expression patterns were displayed: 1) genes that are slowly increased during the healing process, such as bone morphogenetic protein 4, runt-related transcription factor 2, and osteocalcin; 2) genes that are upregulated at the early stage of healing and then downregulated at later stages, such as interleukin and chemokine (C-X-C motif) ligands 2 and 5; and 3) genes that are constitutively expressed over time, such as scleraxis. Although some similarities between osseointegration and tooth extraction socket were seen, distinct features developed and triggered a characteristic coordinated expression and orchestration of transcription factors, growth factors, extracellular matrix molecules, and chemokines. CONCLUSIONS: Characterization of these events contributes to a better understanding of cooperative molecular dynamics in alveolar bone healing, and highlights potential pathways that could be further explored for the enhancement of osseous regenerative strategies.

Organellar proteomics: analysis of pancreatic zymogen granule membranes.

The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.