Human Dental Pulp Fibroblasts Research Articles

Growth hormone and insulin-like growth factor I induce bone morphogenetic proteins 2 and 4: a mediator role in bone and tooth formation?

GH is known to increase the formation of bone and hard tissues of the tooth (dentine, cementum, and enamel), as do bone morphogenetic proteins. GH receptors are expressed in these tissues and could mediate local growth responses. Here we report that both GH and insulin-like growth factor I (IGF-I) are able to increase expression of bone morphogenetic protein-2 and -4 messenger RNAs 4- to 5-fold in human dental pulp fibroblasts in vitro. Induction was seen at physiological concentrations of hormone (25-100 ng/ml GH; 50-200 ng/ml IGF-I) and reached a maximum at 4-8 h. Immunoblot analysis demonstrated that the increase in messenger RNAs resulted in an increase in expressed protein. Anti-IGF-I inhibition experiments indicate that GH is able to induce the response without a requirement for local IGF-I production. These results raise the possibility that bone morphogenetic proteins mediate the local osteogenic actions of GH and IGF-I, and lend support to the view that GH can act through the mediation of factors other than IGF-I. These factors may combine with IGF-I in different tissues to enhance GH action and specificity.

Interleukin-1α stimulates interstitial collagenase gene expression in human dental pulp fibroblast

Interstitial collagenase (matrix metalloprotease-1) is a member of a family of matrix metalloproteases and is thought to play a role in degradation of the extracellular components, such as collagens in normal extracellular matrix remodeling and in many disease processes. We examined interstitial collagenase mRNA expression in human dental pulp fibroblast cultures by Northern blot analysis. These cells did not express interstitial collagenase mRNA in an unstimulated condition. Inflammatory cytokines, such as interleukin-1 alpha, induced interstitial collagenase mRNA expression in these cells. The interstitial collagenase mRNA levels began to increase after 2-h exposure, reaching a maximum after 8 h, then dropping to the unstimulated level at 48 h. These effects were observed in a dose-dependent manner in a dose range of 0.1 to 10 ng/ml. Transforming growth factor-beta reduced the levels of interstitial collagenase mRNA expression that were induced by interleukin-1 alpha. These observations suggest that interstitial collagenase mRNA expression in human dental pulp fibroblasts is regulated by the inflammatory cytokines and that interstitial collagenase may play a role in tissue degradation in inflamed dental pulp.

Bradykinin and thrombin synergistically potentiate interleukin 1 and tumour necrosis factor induced prostanoid biosynthesis in human dental pulp fibroblasts

The effects of and interactions between the six inflammatory mediators interleukin-1 (IL-1), tumour necrosis factor (TNF), gamma-interferon (INF-gamma), transforming growth factor-beta (TGF-beta), bradykinin (BK) and thrombin on prostanoid biosynthesis in primary cultures of human, dental, pulp fibroblasts were examined. IL-1 alpha, IL-1 beta, TNF-alpha and TNF-beta caused a time- and concentration-dependent enhancement of prostaglandin E2 (PGE2) formation in the fibroblasts. The onset of action was delayed 1-2 h and maximal response was seen after 24 h. In contrast, BK and thrombin caused a burst of PGE2 formation with maximal response after 10 min. BK (1 microM) and thrombin (3 U/ml) synergistically potentiated IL-1 alpha and IL-1 beta stimulated PGE2 formation in 24 h cultures. The effect of BK and thrombin on IL-1 beta enhanced PGE2 formation was seen both at suboptimal and optimal concentrations of IL-1 beta without affecting the sensitivity to IL-1 beta. BK and thrombin also synergistically potentiated the stimulatory effect of TNF-alpha and TNF-beta on PGE2 formation. The synergistic interactions between BK and IL-1 alpha, IL-1 beta and TNF-alpha were seen after 2-4 h of treatment. BK analogues with affinity to BK B2-receptors, but not to BK B1-receptors, were able to synergistically potentiate IL-1 beta and TNF-alpha-enhanced PGE2 production. The synergistic stimulation of PGE2 formation by IL-1, TNF and BK was abolished by indomethacin and flurbiprofen. Preincubation with IL-1 beta and TNF-alpha for 24 h resulted in a substantial amplification of the PGE2 response to a subsequent 24 h challenge with BK in the absence of cytokine. Similarly, when the pulp fibroblasts were preincubated with or without IL-1 beta or TNF-alpha for 24 h and then challenged with exogenous arachidonic acid for 60 min, PGE2 formation was significantly enhanced in cytokine pretreated cells. BK potentiated cytokine induced amplification of the PGE2 response to arachidonic acid. gamma-IFN and TGF-beta did not enhance PGE2 formation, nor did these cytokines potentiate IL-beta or TNF-alpha-induced PGE2 production. These data show that proinflammatory mediators such as BK and thrombin act in concert with IL-1 and TNF in stimulating prostanoid formation in human pulpal fibroblasts and that the action of BK is linked to BK B2-receptors. The results are compatible with the view that enhanced metabolism of arachidonic acid, probably due to increased activation or de novo synthesis of cyclooxy-genase(s), is involved in the mechanism by which IL-1, TNF, BK and thrombin interact.

Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide

Interleukin (IL)-8 mRNA expression was investigated in human dental pulp fibroblast cultures after stimulation with lipopolysaccharide (LPS) prepared from Prevotella intermedia and inflammatory cytokines. The expression of IL-8 mRNA and the release of IL-8 induced by P. intermedia LPS in pulpal fibroblast cultures were detected by Northern blot analysis and ELISA, respectively. The sufficient concentration of P. intermedia LPS on the IL-8 mRNA expression was 0.1 microgram/ml in pulpal fibroblast cultures. IL-8 mRNA levels began to increase after 2 h of exposure, reached a maximum at 4 to 8 h, and declined after 48 h, reaching the unstimulated level by 60 h. IL-8 production by the pulpal fibroblasts began to increase after 8 h of exposure upon stimulation with 10 microgram/ml of P. intermedia LPS. By contrast Salmonella LPS and synthetic lipid A did not increase IL-8 mRNA concentrations in pulpal fibroblast cultures. Recombinant human IL-1 alpha, beta, and tumor necrosis factor-alpha were capable of stimulating these cells to express IL-8 mRNA but natural human interferon-beta, gamma, and recombinant human IL-6 were incapable in our assay. These results suggest that pulpal fibroblasts are immunoresponsive cells and can elaborate IL-8 upon stimulation with P. intermedia LPS.

Donor variability in the proliferation of human dental pulp fibroblasts

Fibroblasts were isolated from human dental pulps of healthy third molars from 49 donors of ages ranging from 17 to 68. Significant variability was noted in the success of obtaining primary cultures from these pulps. Variability between the various cultures was also observed in the reliability of maintaining subcultures of the primary cultures as well as recovery from frozen stocks of established cell lines. Of the original 49 explant cultures studied, only three survived long-term passage and freezing. In addition to difficulties and variability in establishing cell lines, the human pulp fibroblasts also showed great variability in proliferative activity which could not be accounted for by donor age, source, or passage number. These findings highlight significant difficulties in establishing reliable human pulp fibroblast cultures and the need for great care in interpreting any in vitro data.

Effects of bradykinin and thrombin on prostaglandin formation, cell proliferation and collagen biosynthesis in human dental-pulp fibroblasts

Bradykinin and thrombin caused a time- and dose-dependent stimulation of prostanoid biosynthesis in human dental-pulp fibroblasts, as assessed by the release of prostaglandin E 2 (PGE 2) and 6-keto-prostaglandin F 1α (the stable breakdown product of prostacyclin). The stimulatory effect of bradykinin and thrombin on PGE 2 biosynthesis was maximal within 5–10 min. The concentration of bradykinin producing half-maximal stimulation (EC 50) of PGE 2 and prostacyclin formation was 10 nM. EC 50 for thrombin-induced formation of PGE 2 and prostacyclin were 0.05 and 0.2 U/ml, respectively. Bradykinin analogues with affinity to the bradykinin B 2 receptor, but not those with affinity to the B 1 receptor, caused a burst of PGE 2 formation. The stimulatory action of bradykinin and thrombin on PGE 2 biosynthesis was abolished by two structurally different cyclo-oxygenase inhibitors and significantly reduced by two corticosteroids. Thrombin dose-dependently enhanced the incorporation of [ 3H]-thymidine into DNA in pulpal fibroblasts by a mechanism that was unrelated to the effect on prostanoid biosynthesis. Bradykinin did not affect thymidine incorporation. Thrombin, but not bradykinin, stimulated the biosynthesis of type 1 collagen in the pulpal fibroblasts. The stimulatory effect of thrombin on collagen biosynthesis was not affected by cyclo-oxygenase inhibitors. These data show that human dental-pulp fibroblasts are equipped with receptors for bradykinin and thrombin linked to enhanced prostanoid biosynthesis. Occupancy of the thrombin receptors also leads to a prostaglandin-independent stimulation of cell proliferation and collagen biosynthesis.

Abstract #65 — Effect of IL-1 on collagen synthesis in human dental pulp fibroblasts

Abstract #65 — Effect of IL-1 on collagen synthesis in human dental pulp fibroblasts

3. Studies with cells derived from growing deer antler

Introduction: The objective of this study was to determine the extent to which substance P (SP) increases proinflammatory cytokine production and osteoclast formation of human dental pulp fibroblasts (HDPF) in patients with severe orthodontically induced inflammatory root resorption (OIIRR). Methods: HDPF were obtained from 5 patients with severe apical root resorption after orthodontic treatment. The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were determined after 24 hours by using ELISA kits. Furthermore, culture supernatants were added to cultured human osteoclasts, and osteoclast formation was observed after tartrate-resistant acid phosphatase (TRAP) staining and the formation of resorption cavities. Results: Stimulation with SP increased the levels of IL-1β, IL-6, and TNF-α, in a time- and concentration-dependent manner, although the increase was greater in the severe root resorption (SRR) group than in the nonresorption (NR) group (P <0.001, 3-way repeated measures ANOVA). As for osteoclast formation, the numbers of TRAP-positive multinucleate cells and resorptive pits were significantly increased in the SRR group compared with the NR group (P <0.001, 2-way repeated measures ANOVA). Conclusions: These results suggest that HDPF stimulated with SP might be deeply involved in the progress of inflammation in pulp tissue and the incidence of SRR during orthodontic treatment.

Supernatant from an Activated Human CD4+ T-Cell Clone Modulates the Proliferation and Collagen Synthesis of Human Dental Pulp Fibroblast

Several studies indicate a relationship between immunocompetent cells and fibroblast functions such as proliferation and collagen synthesis, which may be of importance in understanding the process of fibrosis. We demonstrate here the activity of supernatant from an activated human CD4+ proliferative T-cell clone (2F1) in stirhulating the proliferation of human dental pulp fibroblasts, while inhibiting their soluble type I collagen secretion in either growing or confluent cultures. Taken together, our results indicate that T-cell derived lymphokines may be of importance in regulating normal dental pulp fibroblast functions.