Effects of a triple antibiotic solution on pulpal dynamics after intentionally delayed tooth replantation in mice.

INTRODUCTION: This study analyzed the detailed biological events underlying pulpal dynamics evoked by 3Mix (the mixture of ciprofloxacin, metronidazole, and minocycline) solution after intentionally delayed tooth replantation because 3Mix improves pulpal healing after tooth injuries. METHODS: The maxillary first molars of 3-week-old mice were extracted and immersed in 3Mix solution for 30 minutes in comparison with phosphate buffered saline (PBS) alone. Cell proliferation, apoptosis, and differentiation were assessed in extracted/replanted teeth during days 0-14 using immunohistochemistry, apoptosis assay, and reverse-transcriptase polymerase chain reaction. RESULTS: 3Mix solution accelerated odontoblast differentiation in the coronal pulp on day 7 and tertiary dentin formation on day 14, whereas the regenerative process was delayed in the PBS group. Cell proliferation and apoptosis occurred in the pulp of the 3Mix group during days 5-7 and subsequently decreased from days 7-14. On day 5, dentin sialophosphoprotein and nestin were first recovered in the 3Mix group, whereas expression levels for alkaline phosphatase, osteopontin, and osteocalcin increased in the PBS group. The expression levels for octamer-binding factor 3/4A and 3/4B reached the maximum level on day 1 and were sharply decreased on day 3 in both groups. High expression levels of Cd11c were first observed in the 3Mix group on day 1 and later at days 5 and 7. CONCLUSIONS: The results suggest that the application of 3Mix may suppress osteoblast differentiation by the migration of dendritic cells to the injury site and via the activation of stem/progenitor cells, resulting in the acceleration of odontoblastlike cell differentiation.

In vitro cytocompatibility of N-acetylcysteine-supplemented dentin bonding agents.

INTRODUCTION: Cytotoxic resin components of dentin bonding agents are known to cause oxidative damage and suppress odontogenic differentiation of dental pulp cells. Because antioxidants were found to protect cells from cytotoxicity of resin monomers in previous studies, we investigated the effect of N-acetylcysteine (NAC) on cytotoxicity and anti-differentiation activity of bonding agents. METHODS: Human dental pulp cells were treated with the extracts of dentin bonding agents (Prime & Bond NT, Adper Single Bond, and Dentin Cement), and then cell viability, alkaline phosphatase (ALP) activity, and matrix mineralization were observed. To assess the effects of NAC, NAC was directly added into culture media or mixed with bonding agents before extraction. Release of NAC from bonding agents was also observed by high-performance liquid chromatography. RESULTS: NAC enhanced ALP activity and mRNA expression of dentin sialophosphoprotein gene, whereas extracts of dentin bonding agents inhibited the induction of ALP activity. When the cells were treated with extracts of the bonding agents, the NAC in the culture media reduced the cytotoxicity of the bonding agents. When NAC was incorporated into bonding agents, a protective effect was only seen for Prime & Bond NT containing more than 1% NAC. The disruption of ALP activity and matrix mineralization in pulp cells was partially reversed by NAC only in Prime & Bond NT-treated cells. High-performance liquid chromatography analysis of NAC showed that the amount of NAC effluxed from Prime & Bond NT was not greater than that effluxed from Adper Single Bond. CONCLUSIONS: NAC was useful for reversing cytotoxicity and anti-differentiation effects of Prime & Bond NT on human dental pulp cells.

Nanostructured alumina-coated implant surface: effect on osteoblast-related gene expression and bone-to-implant contact in vivo.

PURPOSE: The use of nanotechnology to enhance endosseous implant surfaces may improve the clinical control of interfacial osteoblast biology. This study investigated the influence of a nanostructure-coated implant surface on osteoblast differentiation and its effects on bone-to-implant contact (BIC) and removal torque values. MATERIALS AND METHODS: Titanium disks were machined (M) or machined and subsequently treated by acid etching (Ac) or by dipping in an aluminum oxide solution (Al2O3). Surfaces were characterized by scanning electron microscopy, atomic force microscopy, and x-ray microanalysis. For the in vitro experiment, rat mesenchymal stem cells (rMSCs) were grown in osteogenic supplements on the disk surfaces for 3 days. Real-time polymerase chain reaction (PCR) was used to measure mRNA levels of several gene products (bone sialoprotein, osteocalcin, osteopontin, and RUNX-2). For the in vivo experiment, titanium implants were placed in rat tibiae and harvested after 3 to 21 days for measurement of bone-specific mRNA levels by real-time PCR. Removal torque and BIC were measured 3 to 56 days after placement. RESULTS: Average height deviation (Sa, in nm) values for M, Ac, and Al2O3 implants were 86.5, 388.4, and 61.2, respectively. Nanostructured Al2O3 topographic features applied to machined implants promoted MSC commitment to the osteoblast phenotype. Greater bone-specific gene expression was observed in tissues adjacent to Al2O3 implants, and associated increases in BIC and torque removal were noted. CONCLUSION: Nanostructured alumina may directly influence cell behavior to enhance osseointegration.

Skeletal developmental effects of selective and nonselective cyclooxygenase-2 inhibitors administered through organogenesis and fetogenesis in Wistar CRL:(WI)WUBR rats.

Cyclooxygenase (COX) inhibitors are the most commonly ingested drugs. The aim of the study was to evaluate the prenatal skeletal effect of selective (DFU) and nonselective (tolmetin, ibuprofen, piroxicam) COX-2 inhibitors. All the tested compounds were administered intragastrically to pregnant Wistar rats from 7 to 21 gestation day. The initial dose was set at 8.5mg/kg/dose for tolmetin and ibuprofen, 0.3 and 0.2mg/kg/dose for piroxicam and DFU. The middle dose was increased 10-times. The highest dose, except for ibuprofen, was elevated 100-times. The highest dose for ibuprofen was set at 200mg/kg/dose. Tolmetin and ibuprofen were administered three times a day. Piroxicam and DFU were dosed once daily. After routine teratological examinations, extremities of randomly selected 21-day-old fetuses were taken for histological, immunohistochemical and molecular studies. The proximal femoral epiphyses were separated and their ultrastructure evaluated. The expression of genes coding cytokines (IL-1alpha, IL-1beta, IL-6, TNF-alpha, TNF-beta) and proteins (COX-1, COX-2, cathepsin K, collagen types I, II and X; osteocalcin, osteopontin) was evaluated in femoral epiphyses by RNase Protection Assay and/or immunohistochemically. The articulate development was checked histologically and found undisturbed in any of the experimental groups. The epiphysis of the 21-day-old fetuses, presented physiological expression of COX-1 and COX-2, as well as cathepsin K, collagen types I, II and X; osteopontin, osteocalcin and TNF-alpha. Increased developmental skeletal variation was noted in groups exposed to the highest dose of nonselective drugs. Unlike the increased number of skeletal variations observed in fetuses exposed to highest doses of nonselective compounds, both groups of COX inhibitors did not disturb joint formation and morphology of femoral epiphyses when administered even in high maternal toxic doses.

L-carnitine and isovaleryl L-carnitine fumarate positively affect human osteoblast proliferation and differentiation in vitro.

Age-related bone loss is characterized by decreased osteoblast activity, possibly related to the reduction of energy production. Carnitine promotes energy availability and its concentration declines with age; Therefore, two Carnitine derivatives, L-carnitine fumarate (LC) and isovaleryl L-carnitine fumarate (Iso-V-LC), have been tested on several parameters of human osteoblasts in vitro. Both compounds significantly increased osteoblast activity, but the new compound Iso-V-LC was more efficient than LC at lower concentrations. They both significantly enhanced cell proliferation, [3H]-proline incorporation and the expression of collagen type I (COLLI), and the bone sialoproteins (BSPs) and osteopontin (OPN). The percentage of alkaline phosphatase (ALP)-positive cells and the secretion of osteocalcin were not modified by LC and Iso-V-LC. Both molecules increased the formation of mineralized nodules, but Iso-V-LC reached the maximum effect at a concentration 10-fold lower than that of LC. Furthermore, we showed that insulin-like growth factor (IGF)-I and IGF-II mRNA levels were not modified by the treatment. However, the two compounds induced an increase of insulin-like growth factor binding protein (IGFBP)-3 and a decrease of IGFBP-5 in both osteoblast lysates and the extracellular matrix (ECM). In conclusion these data suggest that carnitine and, in particular, its new derivative, Iso-V-LC supplementation in the elderly may stimulate osteoblast activity and decrease age-related bone loss.

High-dose retinoic acid modulates rat calvarial osteoblast biology.

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.

IL-1beta-dependent neurological effects of the whole cell pertussis vaccine: a role for IL-1-associated signalling components in vaccine reactogenicity.

Immunization with the whole cell pertussis vaccine (Pw), but not the acellular pertussis vaccine (Pa), is associated with a number of neurological side effects. Previously, we have demonstrated a role for interleukin-1beta (IL-1beta) in Pw reactogenicity. Here we report that parenteral Pw administration resulted in a concomitant increase IL-1 type I receptor (IL-1RI) mRNA and a decrease in IL-1 type II receptor (IL-1RII) mRNA expression in the murine hypothalamus. These Pw-induced changes were accompanied by an increase in caspase-1 and interleukin-1beta (IL-1beta), and were associated with increased activity of the stress-activated kinase, p38. In contrast, immunization with Pa failed to activate pro-inflammatory IL-1 responses but resulted in increased IL-1 receptor antagonist (IL-1ra) production. These results suggest that the neurological effects of Pw are associated with central activation of IL-1beta and IL-1-associated signalling components.