Biological properties versus solubility of endodontic sealers and cements.

Endodontic sealers and cements used in root canal treatment have different compositions and properties. Common to all materials is that their primary goal is to fill gaps and voids, making a permanent seal of the root canal system. Furthermore, aspects such as antibacterial properties, cytotoxicity, setting time, solubility and biocompatibility are also crucial and ought to be considered. Over the years, a shift in the view on the importance of these aspects has ocurred. Whereas the antibacterial properties were considered important when the technical factors in endodontics were less developed, the sealing ability and biocompatibility have later been considered the most critical factors. The introduction of tricalcium silicate cements and sealers has led to a renewed interest in material properties, as these cements seem to have good sealing ability and at the same time combine favourable antimicrobial effects with excellent biocompatibility. This review discusses how the various properties of root canal sealers and cements may conflict with the primary aim of providing a permanent seal of the root canal system.

Differential Responses of Human Dental Pulp Stromal Cells to Bioceramic Materials: A Comparative In Vitro Study.

AIM: The aim of this study was to compare the effects of white MTA-Angelus (wMTA), Biodentine® (Biodentine) and TotalFill® BC Root Repair MaterialTM putty (TotalFill) on human dental pulp stromal cells (hDPSCs) in vitro. MATERIALS AND METHODS: hDPSCs were isolated from third molars of healthy young adults. Material elutes at different concentrations were prepared. Cells were exposed to the eluates for 1, 3, and 7 days. Cell proliferation was evaluated using 3-(4,5-dimethyl-thiazoyl)-2, 5-diphenyl-tetrazolium bromide assay. The expression of alkaline phosphatase (ALP), osteoprotegerin (OPG), osteocalcin (OC), collagen1A (Col1A), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-1 (FGF-1), interleukin 6 (IL6), tumor necrosis factor alpha (TNFα), and interleukin-1-beta (IL1ß) was determined by reverse transcription-polymerase chain reaction (RT-PCR). VEGF-A protein levels and ALP activity were quantified in the culture supernatant. Data were analyzed by two-way analysis of variance (ANOVA). p values <0.05 were considered statistically significant. RESULTS: hDPSC proliferation was decreased in a dose-related manner for all materials on day 3. The same effect was observed with wMTA and TotalFill on day 7. RT-PCR showed that Biodentine increased the expression of the osteogenic markers ALP, OPG, and OC. TotalFill decreased the ALP expression and activity, enhanced the production of angiogenic VEGF-A, and downregulated the inflammatory IL6 on day 7. CONCLUSION: Although the tested materials are used interchangeably in vital pulp therapy, the findings showed varied hDPSC responses. Biodentine did not affect cell proliferation and increased the expression of osteo-/odontogenic markers compared to wMTA and TotalFill, whereas TotalFill decreased cell proliferation and exhibited enhanced angiogenic and anti-inflammatory effects over time. CLINICAL SIGNIFICANCE: The clinical significance of the results needs further investigation in an attempt to provide recommendations on the selection of bioceramic pulp capping material under different scenarios of pulpal pathosis.

Comparison of bone regenerative capacity of donor-matched human adipose-derived and bone marrow mesenchymal stem cells.

Adipose-derived stem cells (ASC) have been used as an alternative to bone marrow mesenchymal stem cells (BMSC) for bone tissue engineering. However, the efficacy of ASC in bone regeneration in comparison with BMSC remains debatable, since inconsistent results have been reported. Comparing ASC with BMSC obtained from different individuals might contribute to this inconsistency in results. Therefore, this study aimed to compare the bone regenerative capacity of donor-matched human ASC and BMSC seeded onto poly(L-lactide-co-ε-caprolactone) scaffolds using calvarial bone defects in nude rats. First, donor-matched ASC and BMSC were seeded onto the co-polymer scaffolds to evaluate their in vitro osteogenic differentiation. Seeded scaffolds and scaffolds without cells (control) were then implanted in calvarial defects in nude rats. The expression of osteogenesis-related genes was examined after 4 weeks. Cellular activity was investigated after 4 and 12 weeks. Bone formation was evaluated radiographically and histologically after 4, 12, and 24 weeks. In vitro, ASC and BMSC demonstrated mineralization. However, BMSC showed higher alkaline phosphatase activity than ASC. In vivo, human osteogenesis-related genes Runx2 and collagen type I were expressed in defects with scaffold/cells. Defects with scaffold/BMSC had higher cellular activity than defects with scaffold/ASC. Moreover, bone formation in defects with scaffold/BMSC was greater than in defects with scaffold/ASC, especially at the early time-point. These results suggest that although ASC have the potential to regenerate bone, the rate of bone regeneration with ASC may be slower than with BMSC. Accordingly, BMSC are more suitable for bone regenerative applications.

Evaluation of Apical Dimension, Canal Taper and Maintenance of Root Canal Morphology Using XP-endo Shaper.

AIM: To evaluate the shaping ability of the XP-endo shaper file system in maxillary molars, representing root canals with variation in morphology. MATERIALS AND METHODS: Twenty maxillary molars were instrumented according to recommended protocols. Pre- and postoperative microcomputed tomography (CT) scans were performed and the root canals classified according to canal type and curvature. The volume change, number of strokes needed to prepare the canals and the size of the 0.04-tapered gutta-percha cone that was adapted in the canal were recorded. RESULTS: XP-endo shaper created a significant change in volume after instrumentation in all the canals, with the biggest change found in the DB canals, followed by the MB1 and the P canals, both at full length and in the apical 4 mm. The number of strokes needed to achieve working length and final shape did not differ between the various root canals. Although not significant, the number of strokes needed to prepare the root canal increased with severity of the curvature, but the severity of the curvature did not result in increased removal of dentin in the apical 4 mm. It was possible to achieve a final root canal size where a 0.04-tapered gutta-percha cone could be adapted. CONCLUSION: The XP-endo shaper was a safe and effective instrument to achieve a root canal preparation of at least size 30 and a 0.04 taper. CLINICAL SIGNIFICANCE: The clinical performance of XP-endo shaper was to some extent dependent on preoperative volume and curvature of the root canal.

Adipose-derived and bone marrow mesenchymal stem cells: a donor-matched comparison.

BACKGROUND: Adipose-derived stem cells (ASCs) have been introduced as an alternative to bone marrow mesenchymal stem cells (BMSCs) for cell-based therapy. However, different studies comparing ASCs and BMSCs have shown conflicting results. In fact, harvesting ASCs and BMSCs from different individuals might influence the results, making comparison difficult. Therefore, this study aimed to characterize donor-matched ASCs and BMSCs in order to investigate proliferation, differentiation potential and possible effects of donor variation on these mesenchymal stem cells (MSCs). METHODS: Human bone marrow and adipose tissue samples were obtained from nine donors aged 8-14. ASCs and BMSCs were isolated and characterized based on expression of surface markers using flow cytometry. The proliferation up to 21 days was investigated. Multi-lineage differentiation was induced using osteogenic, chondrogenic and adipogenic differentiation media. Alkaline phosphatase (ALP) activity was monitored and collagen type I formation was evaluated by immunofluorescence staining. In vitro multi-potency was studied using tissue-specific stains and lineage-specific gene expression. In addition, the osteogenic lineage was evaluated at protein level. RESULTS: Isolated ASCs and BMSCs from all donors demonstrated morphologic and immunophenotypic characteristics of MSCs, with expression of MSCs markers and negative expression of hematopoietic markers. Unlike BMSCs, ASCs showed high expression of CD49d and low expression of Stro-1. In general, ASCs showed significantly higher proliferation and adipogenic capacity with more lipid vesicle formation and expression of the adipogenesis-related genes than BMSCs. In contrast, BMSCs showed significantly higher osteogenic and chondrogenic capacity compared to ASCs. BMSCs had earlier and higher ALP activity, calcium deposition, and expression of the osteogenesis- and chondrogenesis-related genes and the osteogenesis-related protein osteopontin. Proliferation and differentiation capacity of ASCs and BMSCs varied significantly among the donors. CONCLUSIONS: ASCs and BMSCs showed tissue-specific differentiation abilities, but with significant variation between donors. The similarities and differences in the properties of ASCs and BMSCs should be taken into consideration when planning stem cell-based therapy.

Conditioned medium from human bone marrow stromal cells attenuates initial inflammatory reactions in dental pulp tissue.

AIM: To evaluate the effect of MSC-conditioned medium (CM) on the secretion of pro- and anti-inflammatory cytokines from dental pulp cells (hDPC) in vitro, and on the gene expression in vivo after replantation of rat molars. MATERIALS AND METHODS: hDPC were cultured in CM for 24 h, and the concentration of interleukin IL-10, IL-4, IL-6, and IL-8, regulated on activation, normal T Cell expressed and secreted (RANTES), and prostaglandin E2 (PGE2 ) in the media were measured by multiplex assay and ELISA, respectively. Expression of cyclooxygenase-2 (COX-2) was also examined by Western blot analysis after 24 h. Left and right maxillary first rat molars (n = 20) were elevated for 2 min and then replanted with or without application of CM into the tooth sockets. Levels of IL-1ß, IL-10, IL-4, IL-6, and IL-8, and tumor necrosis factor-alpha (TNF-α) mRNA were evaluated by real-time qRT-PCR 3 and 14 days following tooth replantation. RESULTS: The production of IL-8, IL-10, and IL-6, RANTES and PGE2 by cells cultured in CM was significantly higher than production by cells cultured in standard medium (DMEM). At day 3 following replantation in vivo, the levels of IL-1ß and IL-6, and TNF-α mRNA were significantly lower in the CM-treated replanted teeth compared with control teeth. Further, at day 3, the IL-6/IL-10 ratio was significantly lower in the CM-treated replanted teeth compared with control. At day 14 following replantation, no differences in the mRNA ratios were detected between the pulp tissues of replanted and control teeth. CONCLUSIONS: These findings indicated that CM promotes secretion of pro- and anti-inflammatory cytokines from hDPCin vitro and attenuates the initial inflammatory response in the rat dental pulp in vivo following tooth replantation.

Conditioned media from hypoxic-cultured human dental pulp cells promotes bone healing during distraction osteogenesis.

Distraction osteogenesis (DO) is a surgical procedure used to correct various skeletal disorders. Improving the technique by reducing the healing time would be of clinical relevance. The aim of this study was to determine the angiogenic and regenerative potential of conditioned media (CMs) collected from human dental pulp cells (hDPCs) grown under different culture conditions. CM collected from cells under hypoxia was used to improve bone healing and the DO procedure in vivo. The angiogenic potentials of CMs collected from hDPCs grown under normoxic (-Nor) and hypoxic (-Hyp) conditions were evaluated by quantitative PCR (VEGF-A, angiopoietin-1, angiopoietin-2, interleukin-6 (IL-6) and CXCL12), ELISA assays (VEGF-A, Ang-2), tube-formation and wound-healing assays, using human umbilical vein endothelial cells. The results demonstrated that hypoxic CM had significantly higher angiogenic potential than normoxic CM. Human fetal osteoblasts (hFOBs) were exposed to CM, followed by alizarin red staining, to assess the osteogenic potential. It was found that CM did not enhance the mineralization capacity of hFOBs. DO was performed in the tibiae of 30 mice, followed by a local injection of 20 µl CM (CM-Nor and CM-Hyp groups) or serum-free DMEM (control group) into the distraction zone every second day. The mice were sacrificed at days 13 and 27. The CM-Hyp treatment revealed a higher X-ray density than the control group (p < 0.05). Our study suggests that the angiogenic effect promoted by hypoxic culture conditions is dependent on VEGF-A and Ang-2 released from hDPCs. Furthermore, CM-Hyp treatment may thus improve the DO procedure, accelerating bone healing. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

Influence of bone marrow stromal cell secreted molecules on pulpal and periodontal healing in replanted immature rat molars.

AIM: To investigate the effect of paracrine factors secreted from human bone marrow stromal cell (BMSC-CM) on pulpal and periodontal healing following immediate replantation of maxillary rat first molars. MATERIAL AND METHODS: Fifty maxillary rat first molars were elevated and replanted after 2 min. The left teeth were replanted without treatment, whereas BMSC-CM was injected into the right socket prior to replantation. Twelve un-operated teeth served as reference teeth. The expression of vascular endothelial growth factor A, alkaline phosphatase, Runt-related transcription factor 2 and osteoclast stimulating factor 1 was studied by real-time reverse transcription polymerase chain reaction at day 3 and 14. The dentin thickness together with Laminin- and PGP 9.5-immunoreactivity were studied after 3, 14 and 90 days. RESULTS: Real-Time qRT-PCR data showed up-regulated expression of ALP mRNA in the socket specimens of conditioned medium treated replanted teeth after 3 days. No morphological differences were found for the expression of Laminin and PGP 9.5 between control and conditioned medium treated replanted teeth. At day 14, external cervical and surface root resorption was found in one BMSC-CM and one control tooth. At 90 days, all control replanted teeth had external cervical and surface root resorptions, whereas only one sample was seen among the conditioned medium treated teeth. At day 90, more extensive dentine formation with narrowing of the pulpal space was observed in the control compared with conditioned medium treated teeth. CONCLUSIONS: The present findings showed that BMSC-CM treatment reduced the number of replanted teeth with external root resorption and resulted in a significant reduction in new dentin formation.

Bone marrow stromal cell paracrine factors direct osteo/odontogenic differentiation of dental pulp cells.

Growth factors play an important role in osteo/odontogenic differentiation of human dental pulp cells (hDPCs). The aim of this in vitro study was to compare the biological effects of recombinant human growth differentiation factor 5 (rhGDF-5) alone and a cocktail of soluble growth factors (conditioned medium) released from human bone marrow mesenchymal stem cells (hBMMSCs) on the morphology, proliferation and osteo/odontogenic differentiation potential of hDPCs. Passage 4 hDPCs were harvested for culture in four different media: (a) DMEM with 10% FBS, (b) odontogenic induction medium (OM), (c) OM plus 500 ng/mL rhGDF-5, and (d) OM plus conditioned medium (CM). Morphological changes at 48 and 120 h were determined by crystal violet staining. The proliferation rates at 3, 24, 48, and 120 h were assayed by MTT. Using real-time reverse transcription-polymerase chain reaction (RT-PCR), the mRNA levels of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), collagen type I (Col 1), Runt-related transcription factor 2 (Cbfa1/Runx2), alkaline phosphatase (ALP), osteocalcin (OC), ß3 tubulin (TUBB3), glial cell-derived neurotrophic factor (GDNF), angiopoietin-1 (Ang1), and vascular endothelial growth factor A (VEGFA), were determined at 2, 5, and 9 days. Protein expression of dental sialoprotein (DSP), DMP1, OC, and TUBB3 was recorded at 5 days, using western blot and immunocytochemistry. The effect of the different culture media on mineralization was determined by ALP staining at day 5 and Alizarin red S staining at days 7 and 14. In response to the different culture media, the shape of the hDPCs varied from spindled to polygonal and cuboidal. CM inhibited the cellular proliferation rate, while rhGDF-5 had no effect at early time points, but promoted cellular proliferation at 120 h of culture. In the CM group, the mRNA levels of Cbfa1/Runx2, Col 1, ALP, VEGFA, Ang1, and TUBB3 decreased and the levels of GDNF and OC increased. The mRNA levels of DSPP and DMP1 were inconsistent at the time points evaluated. The staining assays also demonstrated that compared with the other groups, the CM group exhibited lower expression of ALP and higher mineralization levels. Protein expression of DSP, DMP1, OC, and TUBB3 was pronounced by the CM-treated cells. It is concluded that under these in vitro conditions, CM released from hBMMSCs have a greater osteo/odontogenic inductive effect on hDPCs than rhGDF-5.

Mesenchymal stem cells induce endothelial cell quiescence and promote capillary formation.

INTRODUCTION: Rapid establishment of functional blood vessels is a prerequisite for successful tissue engineering. During vascular development, endothelial cells (ECs) and perivascular cells assemble into a complex regulating proliferation of ECs, vessel diameter and production of extracellular matrix proteins. The aim of this study was to evaluate the ability of mesenchymal stem cells (MSCs) to establish an endothelial-perivascular complex in tissue-engineered constructs comprising ECs and MSCs. METHODS: Primary human ECs and MSCs were seeded onto poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) scaffolds and grown in dynamic culture before subcutaneous implantation in immunocompromised mice for 1 and 3 weeks. Cellular activity, angiogenic stimulation and vascular assembly in cell/scaffold constructs seeded with ECs or ECs/MSCs in a 5:1 ratio was monitored with real-time RT-PCR, ELISA and immunohistochemical microscopy analysis. RESULTS: A quiescent phenotype of ECs was generated, by adding MSCs to the culture system. Decreased proliferation of ECs, in addition to up-regulation of selected markers for vascular maturation was demonstrated. Baseline expression of VEGFa was higher for MSCs compared with EC (P<0.001), with subsequent up-regulated VEGFa-expression for EC/MSC constructs before (P<0.05) and after implantation (P<0.01). Furthermore, an inflammatory response with CD11b+cells was generated from implantation of human cells. At the end of the 3 week experimental period, a higher vascular density was shown for both cellular constructs compared with empty control scaffolds (P<0.01), with the highest density of capillaries being generated in constructs comprising both ECs and MSCs. CONCLUSIONS: Induction of a quiescent phenotype of ECs associated with vascular maturation can be achieved by co-seeding with MSCs. Hence, MSCs can be appropriate perivascular cells for tissue-engineered constructs.

Endothelial microvascular networks affect gene-expression profiles and osteogenic potential of tissue-engineered constructs.

INTRODUCTION: A major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene-expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation. METHODS: MSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining. RESULTS: The results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks. CONCLUSIONS: This study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs.

Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells.

To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM) was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering.

Systemic chemical desensitization of peptidergic sensory neurons with resiniferatoxin inhibits experimental periodontitis.

BACKGROUND AND OBJECTIVE: The immune system is an important player in the pathophysiology of periodontitis. The brain controls immune responses via neural and hormonal pathways, and brain-neuro-endocrine dysregulation may be a central determinant for pathogenesis. Our current knowledge also emphasizes the central role of sensory nerves. In line with this, we wanted to investigate how desensitization of peptidergic sensory neurons influences the progression of ligature-induced periodontitis, and, furthermore, how selected cytokine and stress hormone responses to Gram-negative bacterial lipopolysaccharide (LPS) stimulation are affected. MATERIAL AND METHODS: Resiniferatoxin (RTX; 50 µg/kg) or vehicle was injected subcutaneously on days 1, 2, and 3 in stress high responding and periodontitis-susceptible Fischer 344 rats. Periodontitis was induced 2 days thereafter. Progression of the disease was assessed after the ligatures had been in place for 20 days. Two h before decapitation all rats received LPS (150 µg/kg i.p.) to induce a robust immune and stress response. RESULTS: Desensitization with RTX significantly reduced bone loss as measured by digital X-rays. LPS provoked a significantly higher increase in serum levels of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α, but lower serum levels of the anti-inflammatory cytokine interleukin (IL)-10 and the stress hormone corticosterone. CONCLUSIONS: In this model RTX-induced chemical desensitization of sensory peptidergic neurons attenuated ligature-induced periodontitis and promoted a shift towards stronger pro-inflammatory cytokine and weaker stress hormone responses to LPS. The results may partly be explained by the attenuated transmission of immuno-inflammatory signals to the brain. In turn, this may weaken the anti-inflammatory brain-derived pathways.

Experimental orthodontic tooth movement and extensive root resorption: periodontal and pulpal changes.

Previous studies have reported changes both in dental pulp and in periodontal ligament (PDL) following orthodontic tooth movement. However, pulpal changes following extensive root resorption after orthodontic tooth movement have not been studied in detail. The aim of this study was therefore to evaluate inflammatory changes, both in the dental pulp and in the compressed PDL, after experimentally induced extensive root resorption. Extensive root resorption was induced in rats by the activation and re-activation of orthodontic force, with a short intervening period of no force application. The distribution of immune cells, nerve fibres and blood vessels was studied immunohistochemically using antibodies against CD68-immunoreactive (IR) cells, major histocompatibility complex (MHC) class II Ia-expressing cells, CD43-IR cells, protein gene product 9.5 (PGP 9.5), and laminin. In the compressed PDL of experimental first molars, significantly increased density of CD68-IR cells and MHC class II Ia-expressing cells were found, whereas the density of CD43-IR cells were unchanged when compared with control second molars. In the compressed PDL, there was an increased density of blood vessels, but no sprouting of nerve fibres. In the dental pulp, however, no increased density of immune cells or sprouting of nerve fibres was recorded. In conclusion, inflammation after extensive root resorption was confined to the compressed PDL, whereas the dental pulp was unaffected.

Importance of lymph vessels in the transcapillary fluid balance in the gingiva studied in a transgenic mouse model.

The gingiva is frequently challenged by oral bacterial products leading to inflammatory responses such as increased fluid filtration and edema formation. The role of initial lymphatics for transcapillary fluid balance in the gingiva is unknown and was therefore investigated in genetically engineered K14-VEGF receptor 3-Ig (K14) lymphedema mice. The mutant mice demonstrated a total lack of lymphatics in the gingiva, whereas lymphatics were found in the submucosal parts of the alveolar mucosa, although they were almost completely absent in the mucosa. In wild-type (WT) mice, lymphatic vessels were detected in mucosal and submucosal parts of the alveolar mucosa. Interstitial fluid pressure (P(if)) measured with micropipettes was increased in the gingiva of K14 mice in the normal situation (P < 0.001) and after inflammation (P < 0.01) induced by lipopolysaccharide from the oral bacteria Porphyromonas gingivalis compared with WT littermates. Fluid volume expansion caused a >75% increase in interstitial fluid volume followed by a drop in P(if) after recovery in both strains. Continuous measurements during the expansion showed an increase in P(if) followed by a decline, suggesting that compliance is increased after the disruption of the extracellular matrix during edema formation. In the alveolar mucosa, no strain differences were observed in P(if) in the normal situation or after fluid volume expansion, suggesting that lymph vessels in the mucosa are not critical for tissue fluid regulation in any situation. Our study demonstrates an important role of gingival lymphatics in transcapillary fluid balance in the steady-state condition and during acute perturbations.

Cytokine signalling in rat pulp interstitial fluid and transcapillary fluid exchange during lipopolysaccharide-induced acute inflammation.

The dental pulp consists of loose connective tissue encased in rigid dentinal walls. Because of its topography the tissue has low interstitial compliance and limited capacity to expand during fluid volume changes. Due to limitations regarding access to interstitial fluid, basic knowledge on transcapillary fluid transport parameters is lacking for this organ. The scope of this project was dual: first we aimed at establishing a method for isolation of pulp interstitial fluid (IF), and second we applied the method in rats subjected to lipopolysaccharide (LPS)-induced endotoxaemia. The aim was to measure colloid osmotic pressure (COP) and pro-inflammatory cytokines in the pulp IF during acute inflammation. Fluid volumes and pulpal blood flow (PBF) were measured to obtain more information about microcirculatory changes that take place in this pulpitis model. By centrifugation of incisor pulp at 239 g we were able to extract fluid representative for IF. Pulp IF had a relative high control COP (approximately 83% of plasma COP) and was similar to plasma COP 3 h after LPS challenge. The pulp exhibited a high content of IF (0.60 +/- 0.03 ml (g wet weight)(-1)) and a vascular volume of 0.03 +/- 0.01 ml (g w.w.)(-1) No differences were observed in the distribution of fluid volumes after 1.5 and 3 h LPS exposure. PBF and systemic blood pressure dropped significantly after LPS administration. PBF remained low whereas systemic blood pressure was re-established during the 3-h period, implying organ dysfunction. There was a differential pattern of cytokine expression in pulp IF and serum with cytokines such as IL-1alpha, IL-1beta and TNF-alpha locally produced, whereas others such as IFN-gamma and IL-6 were produced systemically and probably spilled over to the pulp IF after LPS exposure. Our findings show that pulp IF can be isolated by centrifugation and that this method is useful when studying fluid balance and extracellular signalling mechanisms in the dental pulp in normal and pathological conditions.

Glial cell line-derived neurotrophic factor (GDNF) from adult rat tooth serves a distinct population of large-sized trigeminal neurons.

Glial cell line-derived neurotrophic factor (GDNF) mediates trophic effects for specific classes of sensory neurons. The adult tooth pulp is a well-defined target of sensory trigeminal innervation. Here we investigated potential roles of GDNF in the regulation of adult trigeminal neurons and the dental pulp nerve supply of the rat maxillary first molar. Western blot analysis and radioactive 35S-UTP in situ hybridization revealed that GDNF in the dental pulp and its mRNAs were localized with Ngf in the coronal pulp periphery, in particular in the highly innervated subodontoblast layer. Retrograde neuronal transport of iodinated GDNF and Fluorogold (FG) from the dental pulp indicated that GDNF was transported in about one third of all the trigeminal dental neurons. Of the GDNF-labelled neurons, nearly all (97%) were large-sized (> or =35 microm in diameter). Analysis of FG-labelled neurons revealed that, of the trigeminal neurons supporting the adult dental pulp, approximately 20% were small-sized, lacked isolectin B4 binding and did not transport GDNF. Of the large-sized dental trigeminal neurons approximately 40% transported GDNF. About 90% of the GDNF-accumulating neurons were negative for the high-temperature nociceptive marker VRL-1. Our results show that a subclass of large adult trigeminal neurons are potentially dependent on dental pulp-derived GDNF while small dental trigeminal neurons seems not to require GDNF. This suggests that GDNF may function as a neurotrophic factor for subsets of nerves in the tooth, which apparently mediate mechanosensitive stimuli. As in dorsal root ganglia both small- and large-sized neurons are known to be GDNF-dependent; these data provide molecular evidence that the sensory supply in the adult tooth differs, in some aspects, from the cutaneous sensory system.

Effect of electrical tooth stimulation on blood flow and immunocompetent cells in rat dental pulp after sympathectomy.

Previous experiments show that nerves have effect on the emigration of immunocompetent cells during acute neurogenic inflammation. The present study aims to determine whether the sympathetic or sensory nerves are responsible for emigration of CD43+ and I-A antigen-expressing cells in the dental pulp after electrical tooth stimulation. Wistar rats were used. Experimental rats (n = 6) had the right superior cervical ganglion removed (SCGx), whereas control rats (n = 6) had sham surgery. Fourteen days later, electrical stimulation of the right maxillary 1st molar was performed in both groups for 20-25 s every 5th min for a total period of 4 h. Changes in pulpal blood flow (PBF) were recorded with a laser Doppler flowmeter. All rats were transcardiacally perfused and processed for immunohistochemistry using antibodies against neuropeptides and immune cells. Intermittent electrical stimulation consistently increased PBF and depleted sympathetic and sensory neuropeptides in the dental pulp. The increase in PBF gradually decreased and approached control values at the end of the 4 h stimulation period. A significant increase in the number of I-A antigen-expressing dendritic cells was found in both the SCGx (P < 0.001) and control rats (P < 0.007). In contrast, tooth stimulation did not increase the number of CD43+ cells in the SCGx rats compared to the unstimulated contralateral control molar. Significantly more CD43+ PMN cells (P < 0.01) were found in the control rats after stimulation. It is concluded that stimulation of sympathetic nerves causes recruitment of CD43+ PMN cells, whereas stimulation of sensory nerves causes emigration of I-A antigen-expressing dendritic cells in the dental pulp.

CGRP1 and NK1 receptors in postnatal, developing rat dental tissues.

There is little evidence that neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) participate in the regulation of tooth development. The aim of this study was to analyse the expression of their respective receptors, neurokinin (NK) 1 and CGRP1 receptor, in postnatal developing rat molars and supporting tissues, thereby localizing the target areas for neuropeptide activity. Mol:WIST rats were killed at 7, 14 and 21 d after birth and upper and lower jaws were processed for immunohistochemistry. At early crown stage (P7), only a few individual cells in the dental follicle were receptor positive. At the onset of root formation (P14), post-secretory ameloblasts, cells in the stratum intermedium, the reduced enamel epithelium and the developing alveolar bone demonstrated both NK1 and CGRP1 receptor immunoreactivity. The CGRP1 receptor sites were occasionally evident on cells in the odontoblast layer. At advanced root development (P21), neuropeptide receptor expression was evident on cells close to the developing dentin, cementum and alveolar bone. These data demonstrate dynamic changes in the localization of NK1 and CGRP1 receptors in developing rat dental tissues and indicate an active role for their ligands in the regulation of crown and root development.

Sympathectomy causes increased root resorption after orthodontic tooth movement in rats: immunohistochemical study.

Accumulating evidence suggests that the sympathetic nervous system modulates inflammatory responses and bone remodeling. We have studied the effects of sympathectomy and orthodontic tooth movement (OTM) on root resorption, immunocompetent cell recruitment, neuropeptide, neurokinin-1 receptor (NK1-R), and interleukin 6 (IL-6) expression. Experimental rats (n=8) had the right superior cervical ganglion surgically removed, whereas control rats (n=6) underwent sham surgery. Three days later, all rats had the right maxillary first molar moved mesially by an orthodontic appliance. The rats were perfused 13 days later, and the right maxillae were processed for immunohistochemistry by using primary antibodies directed against ED1 antigen, CD43, substance P (SP), NK1-R, neuropeptide Y (NPY), and IL-6. Following OTM, sympathectomized (SCGx) rats had significantly more root resorption (P<0.01) and SP-immunoreactive (IR) fibers (P=0.01) in the compressed periodontal ligament (PDL) compared with control rats. There was a significant decrease in recruitment of CD43+ cells in the pulp after OTM in SCGx rats compared with control rats (P=0.02). An upregulation of NK1-R immunoreactivity was observed surrounding the hyalinized tissue, and an increase in the number of NK1-R IR cells and density of SP-IR fibers was present in first molar pulp of all rats. NPY-IR fibers were absent in the compressed PDL of SCGx and control rats. Thus, OTM induces remodeling not only around the periodontal tissues, but also in the dental pulp. The sympathetic nerves have an inhibitory effect on hard tissue resorption and a stimulatory effect on CD43+ cell recruitment after OTM.