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Objectives To investigate how scaling affects the penetration of microorganisms into dentinal tubules, how pulpal cells seeded into the pulp cavity respond to bacterial challenge, and how penetration and inflammatory response may depend on the bacterial composition. Materials and methods Root canals of 102 extracted human teeth underwent shaping and cleaning. Half of the teeth were subjected to scaling and root planing, the other half remained untreated. Teeth were exposed to either Streptococcus gordonii and Actinomyces oris or S. gordonii and Porphyromonas gingivalis for 10 weeks. Bacterial invasion was assessed in a depth of 1 mm to the root surface. Human pulpal cells were seeded into the cavities to assess the expression of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and matrix metalloproteinase-3 (MMP-3) by real-time polymerase chain reaction and immunoassay. Results The percentage of teeth with bacteria detected in dentine was higher when teeth received scaling than when they were untreated: 66.6% versus 44.4% when exposed to A. oris/S. gordonii, and 50% versus 25% when exposed to P. gingivalis/S. gordonii (p = 0.043). Scaling had no impact on IL-8 and MMP-3 expression in pulpal cells. P. gingivalis/S. gordonii caused higher levels of IL-8, MCP-1, and MMP-3 than A. oris/S. gordonii (p = 0.003, p = 0.011, p = 0.037). Conclusion Scaling supports the penetration of bacteria into the dentine of extracted human teeth. P. gingivalis may affect the immune response in pulpal cells. Clinical relevance Root surface debridement with hand instruments may facilitate bacterial penetration. Other kinds of mechanical instrumentation in this experimental setting should be investigated.

OBJECTIVES Saliva has been implicated to support oral wound healing, a process that requires a transient inflammatory reaction. However, definitive proof that saliva can provoke an inflammatory response remained elusive. MATERIALS AND METHODS We investigated the ability of freshly harvested and sterile-filtered saliva to cause an inflammatory response of oral fibroblasts and epithelial cells. The expression of cytokines and chemokines was assessed by microarray, RT-PCR, immunoassays, and Luminex technology. The involvement of signaling pathways was determined by Western blot analysis and pharmacologic inhibitors. RESULTS We report that sterile-filtered whole saliva was a potent inducer of IL-6 and IL-8 in fibroblasts from the gingiva, the palate, and the periodontal ligament, but not of oral epithelial cells. This strong inflammatory response requires nuclear factor-kappa B and mitogen-activated protein kinase signaling. The pro-inflammatory capacity is heat stable and has a molecular weight of

The basic function of toothpastes is biofilm removal in order to prevent caries and gingivitis. Toothpastes should provide maximal fluoride availability, optimal abrasivity, and ingredients that do not interfere with fluoride release but should have additional beneficial effects. Further, the effect on cells of the oral cavity is of the utmost importance. We investigated several biological parameters of a new toothpaste (AirFlow-AF) that contains fluoride, xylitol and erythritol but no sodium lauryl sulfate and compared them to commercially available toothpastes (Zendium-Ze, Sensodyne-Se, OdolMed-OM, OralB-OB). The half lethal concentration (LC50) as well as the proliferation behavior on gingival (GF), periodontal ligament (PDL), and mouse fibroblast cells (L929) were was tested. The mean LC50 values of AF on GF, PDL, and L929 were 16.2, 10.9, and 9.3, respectively. In comparison, the four other toothpastes showed mean LC50 values of 1.5 (OB), 1.2 (OM), 1.4 (Se), and 27.7 (Ze) on GF. Mean LC50 values on PDL and L929 were 1.0 and 0.2 (OB), 3.7 and 0.9 (OM), 1.2 and 0.6 (Se), and 25.4 and 5.6 (Ze), respectively. Proliferation behavior mainly confirmed the LC50 values. While cells after stimulation with AF returned to almost unimpaired proliferation behavior at 6%, cells were still strongly impaired after stimulation with all tested commercially toothpastes. AF showed high biocompatibility with different cell types.

The formation of a physiological biofilm cannot be avoided under normal circumstances. However, the consequences of a supragingivally located biofilm, such as caries, gingivitis and, as a further effect, periodontitis, are relatively easy to avoid. The simplest and most common method used worldwide for the elimination of biofilm is periodic mechanical removal using a toothbrush or similar tools, such as chewing sticks or woods. This method was already used in ancient Egypt, and is still being used today, albeit advanced and improved with the help of toothpastes. Here we give a summary of the most common toothbrushes, highlighting their advantages and disadvantages. Furthermore, we provide an overview of the most common toothpastes, their ingredients, and functions. In addition, the ingredients will be critically evaluated and recommendations given for the use or non-use of certain ingredients for different target groups, such as children, healthy adults, or patients with special needs.

The formation of a physiological biofilm cannot be avoided under normal circumstances. However, the consequences of a supragingivally located biofilm, such as caries, gingivitis and, as a further effect, periodontitis, are relatively easy to avoid. The simplest and most common method used worldwide for the elimination of biofilm is periodic mechanical removal using a toothbrush or similar tools, such as chewing sticks or woods. This method was already used in ancient Egypt, and is still being used today, albeit advanced and improved with the help of toothpastes. Here we give a summary of the most common toothbrushes, highlighting their advantages and disadvantages. Furthermore, we provide an overview of the most common toothpastes, their ingredients, and functions. In addition, the ingredients will be critically evaluated and recommendations given for the use or non-use of certain ingredients for different target groups, such as children, healthy adults, or patients with special needs.

Objectives Matrix metalloproteinases (MMPs) in dentin and saliva can degrade collagen. Divalent metals are known inhibitors of MMPs, but stannous – such as in the form of stannous chloride (SnCl2) or stannous fluoride (SnF2) – is yet to be tested for a possible inhibitory effect. In this study, we tested the inhibitory effect on the proteolytic activity of MMP-2 and MMP-9. Methods Sodium chloride (NaCl), sodium fluoride (NaF), and chlorhexidine (CHX) were used as controls. Gelatin zymography was performed with recombinant human MMP-2 and MMP-9. SnCl2, SnF2, NaF, NaCl, and CHX were included either in the incubation buffer (M1) or added to the recombinant MMPs (M2) before the MMPs were analyzed using zymography. Furthermore, the effect of SnCl2, SnF2, and NaF on the enzymatic activity of MMP-2 and MMP-9 was measured in human dentin either before or after acid etching using 37%phosphoric acid. The effect of SnCl2, NaF, and CHX on the viability and of SnCl2 and NaF on the proliferation of human gingival fibroblasts and L929 mouse fibroblasts was also determined. Results For M1, inhibitory concentrations (w/v%) of SnCl2 0.5% and 0.5%, SnF2 0.25% and 0.12%, NaF 0.12% and 0.5%, CHX 0.012% and 0.05%, were observed for MMP-2 and MMP-9, respectively. NaCl had no inhibitory effect. For M2, SnCl2 0.007% and 0.12%, and SnF2 0.03% and 0.5%, inhibited MMP-2 and MMP-9, respectively. NaF, NaCl and CHX had no effect. The enzymatic activity was slightly reduced when SnCl2 and NaF were applied on dentin before the acid attack. Regarding cell viability and proliferation of the cells after stimulation with the respective substances, NaF showed almost no effect, SnCl2 appeared to increase viability and proliferation of the cells, and CHX decreased the viability of cells. Conclusions Stannous ions caused a direct inhibition of the matrix metalloproteinases, whereas F− only had an inhibitory effect when added to the zymography buffer. Clinical significance Inhibition of MMPs using SnCl2 and SnF2 could play an important role in the prevention of dental erosion and caries. However, the clinical relevance of these findings needs to be proven.

The aim of this study was to evaluate the effect of bleaching agents containing different concentrations of hydrogen peroxide (HP) on color-change and on enamel-surface in bovine teeth. Furthermore the influence on cell viability and proliferation was investigated. Two hundred and forty teeth were randomly assigned into four groups (home bleaching ≤6%, in-office bleaching ≤6%, in-office bleaching > 6% HP, and control group). Bleaching was performed after artificial staining and the bleached index (BI) as well as the whiteness index (WID) was measured at several time points. Chemical analysis for HP concentrations and the pH of the bleaching products was done. Furthermore, enamel surfaces of randomly selected specimens were analyzed using scanning electron microscopy (SEM) and cytotoxicity of the tested bleaching products was evaluated in vitro using dental pulp cells (DPCs) and L929 cells. A statistically significant whitening effect was observed in almost all products. As expected all investigated products resulted in decreased cell viability, however, with different values of LC50 (median lethal concentration). SEM analysis showed an analog of enamel alterations with decreasing pH, increasing exposure time, and increasing HP concentration. Bleaching agents containing a low HP concentration are considered to be effective and to have less damaging effects on enamel and tested cells.

Background Development in guided tissue regeneration requires biomaterial testing. 3D cell constructs represent a new approach to bridge the gap between cell culture and animal models. Following the hypothesis that attachment behavior of cells could be observed in toroidal 3D cell constructs, the aim of this study was to evaluate 3D gingival fibroblast (GF) toroids as a simple and feasible in vitro assay to test attachment of oral fibroblasts to collagen membranes. Methods 3D ring-like structures (toroids) were formed from human GF. Hematoxylin-eosin staining was performed with formed GF toroids. Produced GF toroids were seeded onto plastic surfaces or collagen membranes. The morphology was documented at 24 h, 48 h and 72 h after seeding with light and fluorescence microscopy. Toroid vitality was assessed at same time points with a resazurin-based toxicity assay. Results GF showed normal morphology in toroid hematoxylin-eosin staining. Over 72 h, GF toroids on plastic surfaces stayed unchanged, while GF toroids on collagen membranes showed dilatation. GF toroids on plastic surfaces and collagen membranes were metabolically active over the observed period. Conclusions Depending on the surface material, 3D GF toroids show different attachment behavior. Thus, GF toroids are suitable as simple assay to study attachment behavior to various biomaterials.

Background A major mediator of angiogenesis is angiogenin, which is expressed in the early phase of healing in oral tissue engineering strategies. It is unclear how angiogenin is regulated in the periodontal tissue. The objective of this study was to reveal the regulation of angiogenin in response to hypoxia and the hypoxia mimetic agent l-mimosine in periodontal fibroblasts. Methods Human fibroblasts of the periodontal ligament (PDLF) and the gingiva (GF) in monolayer and spheroid cultures were exposed to hypoxia or l-mimosine. The production of angiogenin was evaluated at mRNA and protein levels with reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Echinomycin, an inhibitor of hypoxia-inducible factor (HIF)-1 activity, was used to test the involvement of HIF-1. Results Our data show that hypoxia and l-mimosine can increase angiogenin mRNA and protein levels in PDLF monolayer cultures. In GF monolayer cultures, we found an increase of angiogenin at the mRNA level in response to hypoxia. The increase of angiogenin can be blocked by inhibition of HIF-1 signaling via echinomycin. In PDLF and GF spheroid cultures, the impact of hypoxia and l-mimosine did not reach the level of significance. Conclusion Hypoxia and the hypoxia mimetic agent l-mimosine can increase the production of angiogenin via HIF-1 signaling in PDLF monolayer cultures but not in spheroid cultures. GF were less sensitive to the impact of hypoxia and l-mimosine. Overall, these results suggest a link between hypoxia, HIF-1 signaling and angiogenin in the periodontium.

Trauma in the oral region and tooth avulsion are a major part of injuries in children, adolescents, and young adults. Established methods used to manage these cases provide mainly repair, but not regeneration. Tissue engineering paves the way for research in the new field of regenerative endodontics to overcome the limits of conservative strategies. This review aims to provide an overview of the available in vitro and in vivo models for dental pulp regeneration. The review covers different in vitro models, including two- and three-dimensional models and organ cultures, comprising both ectopic and orthotopic approaches. These models show the great potential of tissue engineering for regenerative endodontics, which can lead to regeneration of the pulp–dentin complex in future.

SUMMARY Objective This study evaluated the differences in enamel color change, surface hardness, elastic modulus, and surface roughness between treatments with four bleaching gels containing carbamide peroxide (two at 10% and one each at 35%, and 45%) and two bleaching gels containing hydrogen peroxide (two at 40%). Methods Enamel specimens were bleached and color changes were measured. Color change was calculated using either ΔE or the Bleaching Index (BI). Then, surface hardness, elastic modulus, and surface roughness of the enamel specimens were evaluated. All measurements were performed at baseline and directly after the first bleaching treatment for all carbamide peroxide– and hydrogen peroxide–containing bleaching gels. In addition, final measurements were made 24 hours after each of a total of 10 bleaching treatments for carbamide peroxide bleaching gels, and 1 week after each of a total of three bleaching treatments for hydrogen peroxide bleaching gels. Results After the last bleaching treatment, respective ΔE scores were 17.6 and 8.2 for the two 10% carbamide peroxide gels, 12.9 and 5.6 for the 45% and 35% carbamide peroxide gels, and 9.6 and 13.9 for the two 40% hydrogen peroxide gels. The respective BI scores were −2.0 and −2.0 for the two 10% carbamide peroxide gels, −3.5 and −1.5 for the 45% and 35% carbamide peroxide gels, and −2.0 and −3.0 for the two 40% hydrogen peroxide gels. Each bleaching gel treatment resulted in significant whitening; however, no significant difference was found among the gels after the last bleaching. Whitening occurred within the first bleaching treatments and did not increase significantly during the remaining treatments. Surface hardness significantly decreased after the last bleaching treatment, when 10% carbamide peroxide was used. Furthermore, significant changes in the elastic modulus or surface roughness occurred only after treatment with 10% carbamide peroxide. Conclusion All six bleaching gels effectively bleached the enamel specimens independent of their concentration of peroxide. Gels with low peroxide concentration and longer contact time negatively affected the enamel surface.

Dentale Erosionen und erosiver Zahnhartsubstanzverlust erlangten innerhalb der letzten Jahre einen immer hoheren Stellenwert fur Zahnarzte, Wissenschaftler und auch Patienten. Dieses zunehmende Interesse ist auf den vorherrschenden Lebensstil, einen hohen Konsum erosionsfordernder Speisen und Getranke sowie auf in der westlichen industrialisierten Gesellschaft haufiger auftretende internistische Gesundheitsprobleme zuruckzufuhren. Dieser Beitrag gibt einen Uberblick uber die heutige Definition, die Entstehung, haufigste Risikofaktoren sowie die Diagnostik, Therapie und Pravention.

PURPOSE Whole saliva comprises components of the salivary pellicle that spontaneously forms on surfaces of implants and teeth. However, there are no studies that functionally link the salivary pellicle with a possible change in gene expression. MATERIALS AND METHODS This study examined the genetic response of oral fibroblasts exposed to the salivary pellicle and whole saliva. Oral fibroblasts were seeded onto a salivary pellicle and the respective untreated surface. Oral fibroblasts were also exposed to freshly harvested sterile-filtered whole saliva. A genome-wide microarray of oral fibroblasts was performed, followed by gene ontology screening with DAVID functional annotation clustering, KEGG pathway analysis, and the STRING functional protein association network. RESULTS Exposure of oral fibroblasts to saliva caused 61 genes to be differentially expressed (P < .05). Gene ontology screening assigned the respective genes into 262 biologic processes, 3 cellular components, 13 molecular functions, and 7 pathways. Most remarkable was the enrichment in the inflammatory response. None of the genes regulated by whole saliva was significantly changed when cells were placed onto a salivary pellicle. CONCLUSION The salivary pellicle per se does not provoke a significant inflammatory response of oral fibroblasts in vitro, whereas sterile-filtered whole saliva does produce a strong inflammatory response.

Calcium hydroxide removal from the root canal by photon induced photoacoustic streaming (PIPS) compared to needle irrigation and irrigation using sonic activation was investigated. Additionally, safety issues regarding apical extrusion were addressed. In endodontic treatment temporary intracanal medication like calcium hydroxide should be completely removed for long term success. For analysis, 60 artificial teeth were prepared, filled with calcium hydroxide, and divided into four groups. The teeth were assigned to needle irrigation, irrigation using a sonic device, PIPS with a lower energy setting (10 mJ, 15 Hz), or PIPS with a higher energy setting (25 mJ/40 Hz). For comparison the weight of each tooth was measured before and after calcium hydroxide incorporation, as well as after removing calcium hydroxide using the four different methods. Regarding safety issues another 24 samples were filled with stained calcium hydroxide and embedded in 0.4% agarose gel. Color changes in the agarose gel due to apical extrusion were digitally analysed using Photoshop. No significant differences were found for calcium hydroxide removal between the two laser groups. Sonic assisted removal and needle irrigation resulted in significant less calcium hydroxide removal than both laser groups, with significantly more calcium hydroxide removal in the ultrasonic group than in the needle irrigation group. For apical extrusion the higher laser (25 mJ/40 Hz) group resulted in significant higher color changes of the periapical gel than all other groups. PIPS with the setting of 10 mJ/15 Hz achieved almost complete removal of calcium hydroxide without increasing apical extrusion of the irrigation solution.

Dental caries is still a global oral health burden decreasing quality of life of affected children and adults. In addition to accepted oral prophylactic techniques fluoride therapy and regular oral hygiene visits, the positive additional benefit of sealing susceptible pits and fissures is rhetorical. Treatment strategies for occlusal but also other carious lesions have shifted more and more from an invasive procedure toward minimal invasive or even non-operative strategies. Sealing non-cavitated occlusal carious lesions is one of these strategies. This chapter is intended to discuss the various strategies of pit and fissure sealing in the management of non-cavitated occlusal carious lesions in order to arrest their progression.

To determine the performance of a resin composite material specially developed for core build-ups in comparison with conventional restorative materials.90 roughened ceramic blocs were divided into three groups; one group (n=30) was used for the core build-up material (Gradia Core) and the other two groups (n=30, each) were used for two conventional restorative materials (Tetric EvoCeram, Compoglass F). After adhesive fixation, specimens of each material were subdivided in accordance with the storage conditions (thermocycling or water storage). Shear bond strength was measured and fracture behavior was analyzed.Gradia Core presented significantly higher shear bond strength values than the conventional restorative material Tetric EvoCeram, both after 24 hours water storage as well as after thermocycling. Compoglass F did not show any statistically significant differences compared to the other materials, independent of the storage condition. However, Compoglass F resulted in numerically higher shear bond values than Tetric EvoCeram, but lower shear bond values than Gradia Core. Within the same materials, no statistically significant differences occurred regarding the storage conditions.The specific core build-up material provided stronger bonding properties when luted to feldspar ceramic than conventional restorative materials, making it a suitable supporting material when high-quality esthetic restorations are needed for restoring decayed, but vital teeth.

Purpose Purpose of this study was to evaluate the accuracy of fit of cemented polymer infiltrated ceramic network (PICN) material crowns manufactured after digital and conventional impression techniques using micro computed tomography (CT). Furthermore to determine the cement space volume and porosities in the cement layer. Methods A molar typodont tooth was prepared for PICN material crowns and replicated thirty times. The dies were randomly divided into three groups of 10 specimens each according to the impression technique: 3M True Definition Scanner (TDS), cara TRIOS (Trios) and Impregum Penta Soft (Impregum). PICN material crowns were milled for each specimen from Vita Enamic blocks and cemented on their respective dies. The absolute marginal discrepancy (AMD), internal fit (IG), total cement space volume (TVC) and marginal porosities (VP) were measured using Micro-CT. Results Mean and standard deviations values in μm for the AMD were: TDS 140.1 (28.4); Trios 253.7 (56.8); Impregum 220.2 (101.1). IG values in μm: TDS 173.1 (27.7); Trios 222.2 (22.4); Impregum 211.6 (55.9). TVC in mm3: TDS 19.82 (2.9); Trios 23.67 (2.01); Impregum 23.77 (5.09). VP in mm3: TDS 0.38 (0.09); Trios 0.36 (0.10); Impregum 0.51 (0.31). Conclusions TDS group showed significantly better marginal and internal fit than the Trios group. No difference of the parameters was detected between the Impregum and both digital groups which implies that the digital impression technique is suitable in the manufacturing process of PICN material crowns.

Background To understand the responses of the dental pulp to hypoxia is of high relevance for regenerative endodontics and dental traumatology. Here, we aimed to reveal the effects of hypoxia and the hypoxia mimetic agent L-mimosine (L-MIM) on the production of sclerostin (SOST) and dickkopf-1 (DKK-1) in human dental pulp-derived cells (DPC). Methods DPC in monolayer, spheroid and tooth slice cultures were treated with L-MIM or hypoxia. Resazurin-based toxicity and MTT assays were performed to determine cell viability. mRNA and protein levels of SOST and DKK-1 were measured with quantitative reverse transcription PCR and ELISA, respectively. To validate the hypoxia-like response, SDF-1, VEGF and IL-8 were assessed. In addition Western blots for HIF-1α, HIF-2α and HIF-3α were done. Results Cells were vital upon treatment procedures and showed increased levels of HIF-1α, and HIF-2α. In monolayer cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM and hypoxia, respectively. A significant downregulation of SOST by hypoxia was found at the protein level compared to untreated cells while the effect on DKK-1 and the impact of L-MIM on SOST and DKK-1 did not reach the level of significance at the protein level. In spheroid cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM. A significant downregulation of DKK-1 upon hypoxia treatment was found at the protein level while the impact of hypoxia on SOST and the effect of L-MIM on SOST and DKK-1 did not reach the level of significance. SOST and DKK-1 were also produced in tooth slices, but no pronounced modulation by L-MIM or hypoxia was found. Evaluation of SDF-1, VEGF and IL-8 showed a hypoxia-like response in the culture models. Conclusions There is no pronounced influence of hypoxia and L-MIM on DPC viability, SOST and DKK-1 protein production. However, the specific response depends on the culture model and the level of evaluation (mRNA or protein). These results deepen our understanding about the role of hypoxia and the potential impacts of hypoxia-based strategies on dental pulp.

Background: Damage or exposure of the dental pulp requires immediate therapeutic intervention. Methods: This study assessed the biocompatibility of a silver-containing PLGA/TCP-nanofabric scaffold (PLGA/Ag-TCP) in two in vitro models, i.e. the material adapted on pre-cultured cells and cells directly cultured on the material, respectively. Collagen saffolds with and without hyaluronan acid (Coll-HA; Coll) using both cell culturing methods and cells growing on culture plates served as reference. Cell viability and proliferation were assessed after 24, 48, and 72 h based on formazan formation and BrdU incorporation. Scaffolds were harvested. Gene expression of interleukin(IL)-6, tumor necrosis factor (TNF)-alpha, and alkaline phosphatase (AP) was assessed 24 h after stimulation. Results: In both models formazan formation and BrdU incorporation was reduced by PLGA/Ag-TCP on dental pulp cells, while no significant reduction was found in cells with Coll and Coll-HA. Cells with PLGA/Ag-TCP for 72 h showed similar relative BrdU incorporation than cells stimulated with Coll and Coll-HA. A prominent increase in the pro-inflammatory genes IL-6 and TNF-α was observed when cells were cultured with PLGA/Ag-TCP compared to the other groups. This increase was parallel with a slight increase in AP expression. Overall, no differences between the two culture methods were observed. Conclusions: PLGA/Ag-TCP decreased viability and proliferation rate of human dental pulp cells and increased the pro-inflammatory capacity and alkaline phosphatase expression. Whether these cellular responses observed in vitro translate into pulp regeneration in vivo will be assessed in further studies., BMC Oral Health, 17, ISSN:1472-6831

To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model.Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test.Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P 0.05). This effect on VEGF was reduced by echinomycin (P 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P 0.05) and IL-8 levels (P 0.05).The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Background Evaluation of the marginal fit of cemented zirconia copings manufactured after digital impression with Lava™ Chairside Oral Scanner in comparison to that of zirconia copings manufactured after conventional impressions with polyvinyl siloxane. Methods A prepared typodont tooth #36, was replicated 40 times with a vinyl silicone and precise model resin. The dies were randomly divided into two groups according to the impression taking technique. Digital impressions with Lava™ C.O.S. and conventional impressions were taken according to the group. Subsequently zirconia copings were manufactured and cemented on their respective dies with zinc oxide phosphate cement. After embedding in resin, mesio-distal section of each coping was performed with a diamond saw in order to obtain two slices. One half of the specimen was used for evaluation with an optical microscope (OM) and the other half for evaluation with a scanning electron microscope (SEM). Marginal gap (MG) and absolute marginal discrepancy (AMD) were measured mesial and distal on each slice. Results No significant difference of the marginal parameters between the digital and the conventional group was found. The mean values for MG in the digital group were 96.28 μm (+/−43.21 μm) measured with the OM and 99.26 μm (+/−48.73 μm) measured with the SEM, respectively. AMD mean values were 191.54 μm (+/−85.42 μm) measured with the optical microscope and 211.6 μm (+/−96.55 μm) with the SEM. For the conventional group the mean MG values were 94.84 μm (+/−50.77 μm) measured with the OM and 83.37 μm (+/−44.38 μm) measured with the SEM, respectively. AMD mean values were 158.60 μm (+/−69.14 μm) for the OM and 152.72 μm (+/−72.36) for the SEM. Conclusions Copings manufactured after digital impression with Lava™ C.O.S. show comparable marginal parameters with the copings manufactured after conventional impression with polyvinyl syloxane. The mean MG values of both groups fit in the clinically acceptable range. Electronic supplementary material The online version of this article (doi:10.1186/s12903-016-0323-8) contains supplementary material, which is available to authorized users.

Introduction Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp–derived cells. Methods To test the response of dental pulp–derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, 3 [H]thymidine, and 3 [H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). Results We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp–derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. Conclusions These results show that PHD inhibitors can stimulate VEGF production of dental pulp–derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Explantation of failed dental implants using a piezoelectric device may provide a viable treatment modality, especially for implants in precarious locations. The traditional mechanical procedures compared to piezosurgical technique for maxillary implant removal are described. Furthermore, the remaining bone on dental implants was analyzed by scanning electron microscopy (SEM) in order to semi-quantitatively evaluate the amount and structure. The piezoelectric device appears to be a valuable minimally invasive alternative if removal of dental implants is required.

The bond joint between dentin and ceramic is a critical determinant in prosthodontic dentistry. The laser is an alternative to the diamond bur for preparing tooth cavities. However, the impact of lasers on the bond between the laser-irradiated dentin and the ceramic remains a matter of controversy. We determined the shear strength of bonds between ceramic blocks and human dentin discs prepared with either an Er,Cr:YSGG laser or a diamond bur. A total of 180 dentin discs were randomly assigned to four groups. Three groups of discs were prepared with the Er,Cr:YSGG laser irradiation (2 W, 30 Hz, 50% H(2)O, 70% air) and the fourth group was prepared with a diamond bur. In one of the laser groups the discs surfaces were also treated with phosphoric acid and in another with phosphoric acid and mechanical smoothing using a dental excavator. The ceramic blocks were bonded to the dentin discs with Syntac adhesive (together with Variolink II curing system), ExciTE adhesive (together with Variolink II curing system) or RelyX self-adhesive cement. The shear strength of the bond between ceramic and dentin was significantly higher following dentin surface treatment with the laser alone than following treatment with the diamond bur and Variolink II/Syntac (p = 0.021) but not significantly higher than following treatment with the diamond bur and Variolink II/ExciTE (p = 0.138) or RelyX (p = 0.150). A significant difference was not observed when the laser-treated dentin was conditioned with phosphoric acid and mechanical smoothing. These findings demonstrate that the bond between dentin and ceramic may be stronger after laser irradiation; however, the selection of the adhesive material is an additional factor that affects the bond strength.

Cvikl B, Agis H, Stogerer K, Moritz A, Watzek G, Gruber R. The response of dental pulp-derived cells to zoledronate depends on the experimental model. International Endodontic Journal, 44, 33–40, 2011. Abstract Aim To investigate whether zoledronate (ZOL) can cause a cytotoxic response in dental pulp-derived cells (DPCs) in vitro. Methodology Cell activity was assessed utilizing MTT tests, 3[H]thymidine, and 3[H]leucine incorporation assays in human DPCs in response to ZOL. Cell activity assays were also preformed on calcium phosphate-coated plates. Cell death was analysed with annexin V/propidium iodide, trypan blue staining and Western blot analysis. Results Micromolar concentrations of ZOL were required to decrease the activity of DPCs. The decreased activity of DPCs was associated with the occurrence of apoptosis and necrosis. No adverse effects were observed when DPCs were cultured on calcium phosphate-coated plates with ZOL. Conclusion High concentrations of soluble ZOL were required to cause adverse effects in vitro. These adverse effects are abolished when the bisphosphonate was bound to a mineralized surface. However, the clinical relevance of these results remains to be determined.

OBJECTIVES Artificial saliva is widely used to overcome reduced natural salivary flow. Natural saliva provokes the expression of chemokines in oral fibroblasts in vitro. However, if artificial saliva changes the expression of chemokines remains unknown. MATERIALS AND METHODS Here, we investigated the ability of Saliva Orthana®, Aldiamed®, Glandosane®, and Saliva Natura® to change the expression of chemokines in human oral fibroblasts and the human oral epithelial cell line HSC-2 by means of reverse transcription polymerase chain reaction and immunoassays. Mucins isolated from bovine submaxillary glands and recombinant human mucin 1 were included in the bioassay. Formazan formation and LIVE/DEAD® staining determined the impact of artificial saliva on cell viability. The involvement of signaling pathways was determined by pharmacologic inhibitors and Western blotting. RESULTS In gingival fibroblasts, Saliva Orthana®-containing mucins provoked a significantly increased expression of CXC ligand 8 (CXCL8, or interleukin 8), CXCL1, and CXCL2. Immunoassays for CXCL8 and CXCL1 confirmed the translation at the protein level. The respective dilution of artificial saliva had no impact on formazan formation and LIVE/DEAD® staining. Mucins isolated from bovine submaxillary glands also increased the panel of chemokine expression in gingival fibroblasts. BAY 11-7082, a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, but also TAK-242, an inhibitor of toll-like receptor 4 signaling, blocked chemokine expression of Saliva Orthana® and bovine mucins. In HSC-2 cells, Glandosane® significantly increased CXCL8 expression. CONCLUSIONS Saliva Orthana® stimulated chemokine expression in gingival fibroblasts. Mammalian mucins, but also possible contaminations with endotoxins, might contribute to the respective changes in gene expression. Epithelial cells have a differential response to artificial saliva with Glandosane® changing CXCL8 expression. CLINICAL RELEVANCE Artificial saliva can incite a cellular response, if however the changing expression of chemokines by isolated fibroblasts and epithelial cells in vitro translates into a clinical condition, is not clear.

Introduction Proangiogenic prolyl hydroxylase (PHD) inhibitors represent a novel approach to stimulate tissue regeneration. Diabetes mellitus involves the accumulation of advanced glycation end products (AGEs). Here we evaluated the impact of AGEs on the response of human pulp tissue to the PHD inhibitor L-mimosine (L-MIM) in monolayer cultures of dental pulp–derived cells (DPCs) and tooth slice organ cultures. Methods In monolayer cultures, DPCs were incubated with L-MIM and AGEs. Viability was assessed based on formazan formation, live-dead staining, annexin V/propidium iodide, and trypan blue exclusion assay. Vascular endothelial growth factor (VEGF), interleukin (IL)-6, and IL-8 production was evaluated by quantitative polymerase chain reaction and immunoassays. Furthermore, expression levels of odontoblast markers were assessed, and alizarin red staining was performed. Tooth slice organ cultures were performed, and VEGF, IL-6, and IL8 levels in their supernatants were measured by immunoassays. Pulp tissue vitality and morphology were assessed by MTT assay and histology. Results In monolayer cultures of DPCs, L-MIM at nontoxic concentrations increased the production of VEGF and IL-8 in the presence of AGEs. Stimulation with L-MIM decreased alkaline phosphatase levels and matrix mineralization also in the presence of AGEs, whereas no significant changes in dentin matrix protein 1 and dentin sialophosphoprotein expression were observed. In tooth slice organ cultures, L-MIM increased VEGF but not IL-6 and IL-8 production in the presence of AGEs. The pulp tissue was vital, and no signs of apoptosis or necrosis were observed. Conclusions Overall, in the presence of AGEs, L-MIM increases the proangiogenic capacity, but decreases alkaline phosphatase expression and matrix mineralization.

AIM: To investigate the inflammatory response of dental pulp fibroblasts and the respective explants to whole saliva. METHODOLOGY: Explants from human and porcine dental pulp tissue and isolated dental pulp fibroblasts were used to investigate the inflammatory response to sterile saliva. Cytokine and chemokine expression was assessed by RT-PCR. Western blot analysis and pharmacologic inhibitors were used to determine the involvement of signalling pathways. RESULTS: Dental pulp explants of human and porcine origin exposed to human saliva exhibited no major changes of IL-6 and IL-8 mRNA expression (P > 0.05). In contrast, isolated porcine and human dental pulp fibroblasts, when stimulated with human saliva, exhibited a vastly increased expression of IL-6 and IL-8 mRNA (P < 0.05). In pulp fibroblasts, saliva also increased the expression of other cytokines and chemokines via activation of NFkappaB, ERK and p38 signalling. Notably, a significantly reduced inflammatory response was elicited when pulp fibroblasts were transiently exposed to saliva. CONCLUSIONS: Saliva has a potential impact on inflammation of dental pulp fibroblasts in vitro but not when cells are embedded in the intrinsic extracellular matrix of the explant tissue.

The ability to generate new bone for reconstruc- tive surgery use is a major clinical need. Tissue engineering with osteoprogenitor cells isolated from the patient's peri- osteum and seeded into bioresorbable scaffolds offers a promising approach to the generation of skeletal tissue. To our knowledge, there is no description about the expression of Ets2 in tissue engineered "bone neotissue." The aim of our study was to manufacture cell-seeded three-dimensional bone constructs with human periosteal cells on poly (lactic- co-glycolic acid) polymer fleeces to describe the expression pattern of Ets2 and its target genes osteocalcin and os- teopontin; expression analysis of type I collagen, core-bind- ing factor-1, alkaline phosphatase, and osteonectin; the abil- ity of matrix mineralization and ALP enzymatic activity showed the osteogenic character of the constructs. A signif- icant correlation between the expression of Ets2 and os- teopontin mRNA (r 0.70; p 0.05) could be shown. A 1.35-fold increase of Ets2 expression from days 1 to 9 was detected, followed by a slight decrease from days 11 to 15. Until the end of the culture period, the expression of Ets2 reached a comparable high level as detected on day 9. In contrast, the expression level of osteopontin mRNA reached a maximum at day 7, followed by a progressive 3.04-fold decrease until day 21. This study shows for the first time that Ets2 gene and its transcriptional target genes are expressed in tissue-engineered bone constructs. These findings have the potential to provide much-needed information about the role and function of Ets2 in human osteogenesis processes and creation of "bone neotissue." © 2005 Wiley Periodicals, Inc. J Biomed Mater Res 73A: 445- 455, 2005

Bone tissue engineering is a promising approach for treatment of defective and lost bone in the maxillofacial region. Creating functional tissue for load bearing bone reconstruction using biocompatible and biodegradable scaffolds seeded with living cells is of crucial importance. The aim of our study was to compare the effects of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) ceramic granulae on growth, differentiation, mineralization and gene expression of mandibular mesenchymal cambial layer precursor cells (MCLPCs) cultured onto tissue engineered three-dimensional (3-D) composites in vitro. These 3-D composites were cultivated in a rotating cultivation system under osteogenic differentiation conditions for a maximum period of 21 days. After 6 and 21 days, histological examination was performed; scanning electron microscopy (SEM), alkaline phosphatase (ALP) activity and levels of DNA were investigated. Expression of bone-specific genes osteocalcin, osteonectin, osteopontin, ALP, core binding factor alpha 1 and collagen type I were investigated by using a reverse transcription-polymerase chain reaction (RT-PCR) method. After 6 and 21 days of incubation an initiation of mineralization and the presence of newly formed bone at the surface of the composites were shown after evaluation of ALP activity, DNA content, SEM and histological staining. Expression of bone-specific genes confirmed the bone-like character of these composites and different effects of PLGA or HA granulae on the osteogenic differentiation of human MCLPCs in vitro. The results of this study support the concept that substrate signals significantly influence MCLPCs growth, differentiation, mineralization and gene expression in vitro, and that the use of these cells in the manufacturing of 3-D cell/HA composites is a promising approach for load bearing bone reconstruction in the maxillofacial region in vivo.

A new approach to addressing difficult tissue reconstructive or replacement problems in the oral cavity is to engineer new tissue by using selective cell transplantation on polymer scaffolds. The current study characterized the osteoblastic nature of adherent mandibular cells on biomaterials, which could have a potential use as scaffolds for tissue engineering strategies. Cells of mandibular origin from one patient were cultivated on three different biomaterials (PepGen P-15 trade mark, Frios Algipore, and OsteoGraf/LD-700) for 7 and 14 days and osteocalcin expression was demonstrated by RT-PCR and SDS-PAGE/Western blotting. In order to explicitly characterize only the adherent cells on the biomaterials, we first separated the biomaterials with adherent cells from the culture plate before trypsinization. We could demonstrate that cell growth of adherent mandibular osteoblast-like cells was significantly higher on biomaterials with an organic component (PepGen P-15 trade mark ) in comparison to Frios Algipore and OsteoGraf/LD-700, respectively. In conclusion, only the explicit study of adherent cells at the gene and protein levels gives information about the osteoconductivity of biomaterials.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Objective: The aim of this study was to investigate the effect of different energy settings of Er:YAG laser irradiation on dentin surface morphology with respect to the number of opened dentinal tubules. Background data: An ideally prepared dentin surface with opened dentinal tubules is a prerequisite for adhesive fixation. No study, however, has yet compared the numbers of opened dentinal tubules with regard to statistical differences. Methods: Conventional preparations using a bur with or without additional acid etching acted as control groups. Dentin specimens were prepared from human third molars and randomly divided into eight groups according to the energy settings of the laser (1, 1.5, 4, 6, 7.5, and 8 W) and two controls (bur and bur plus acid etching). After surface preparation, dentin surfaces were analyzed with a scanning electron microscope, and the number of opened dentinal tubules in a defined area was counted. Results: The control groups showed smooth surfaces with (bur plus acid etching) and without opened dentinal tubules (bur), whereas all laser-irradiated surfaces showed rough surfaces. Using the energy setting of 4 W resulted in significantly more opened dentinal tubules than the conventional preparation technique using the bur with additional acid etching. In contrast, the energy setting of 8 W showed significantly fewer opened dentinal tubules, and also exhibited signs of thermal damage. Conclusions: The Er:YAG laser with an energy setting of 4 W generates a dentin surface with opened dentinal tubules, a prerequisite for adhesive fixation.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86—all costimulatory molecules of phagocytic cells—were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

Fractured endodontic instruments inhibit optimal cleaning and filling of dental root canals, which may result in a less favorable prognosis for the tooth. Several techniques are available to remove fractured instruments; however, healthy tooth substance often must be destroyed in the process. This study was intended to evaluate Nd:YAG laser treatment as a method to remove fractured stainless steel instruments without destroying healthy tooth substance.Stainless steel endodontic instruments were fractured in 33 unprocessed root canals of mandibular central and lateral incisors and premolars in vitro. A brass tube charged with solder was placed at the coronal end of the fractured instrument and laser energy was used to melt the solder, connecting the fractured instrument with the brass tube. The success rates of connecting and removal of fractured instruments from the root channel were recorded for each case.Connecting was achieved in every case in which more than 1.5 mm of the fractured instrument was tangible (22 out of 22). In cases where less than 1.5 mm was tangible, the rate for successful connection decreased to 4 out of 11 (36.4%). Fractured endodontic instruments were removed successfully in 17 out of 22 cases (77.3%) in which more than 1.5 mm was tangible. If less than 1.5 mm was tangible, the removal success rate decreased to 3 out of 11 cases (27.3%).Our data support Nd:YAG laser-mediated connecting of a brass tube to a fractured endodontic instrument as a feasible and tissue conserving removal approach when more than 1.5 mm of the instrument is tangible.

To determine the best-performing combination of three core buildup materials and three bonding materials based on their bond strength to ceramic blocks in vitro.The materials used for core buildup were a composite (Tetric EvoCeram), a compomer (Compoglass F), and a glass-ionomer cement (Ketac Fil Plus), and for bonding, a three-step etch-and-rinse adhesive (Syntac), a two-step etch-and-rinse adhesive (ExciTE), and a single-step system (RelyX Unicem). Bond strength to ceramic blocks was determined by shear bond strength testing. Fracture behavior was evaluated by scanning electron microscopy.The highest adhesive values between buildup and ceramic were obtained using the materials Compoglass F and Syntac, followed by Compoglass F and ExciTE. Among the two other core buildups, Tetric EvoCeram performed better than Ketac Fil Plus, which was independent of the bonding materials. Adhesive fractures were characteristically observed with Syntac and ExciTE, and cohesive fractures were characteristically observed with RelyX Unicem.These data show that compomers bonded with a multistep adhesive system achieved statistically significantly higher shear bond strength than composites and glass-ionomer cements. Within the limitations inherent to this in vitro study, the use of compomers for core buildup can be recommended.

Bone tissue engineering is a promising approach for treatment of defective and lost bone in the maxillofacial region. Creating functional tissue for load bearing bone reconstruction using biocompatible and biodegradable scaffolds seeded with living cells is of crucial importance. The aim of our study was to compare the effects of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) ceramic granulae on growth, differentiation, mineralization and gene expression of mandibular mesenchymal cambial layer precursor cells (MCLPCs) cultured onto tissue engineered three-dimensional (3-D) composites in vitro. These 3-D composites were cultivated in a rotating cultivation system under osteogenic differentiation conditions for a maximum period of 21 days. After 6 and 21 days, histological examination was performed; scanning electron microscopy (SEM), alkaline phosphatase (ALP) activity and levels of DNA were investigated. Expression of bone-specific genes osteocalcin, osteonectin, osteopontin, ALP, core binding factor alpha 1 and collagen type I were investigated by using a reverse transcription-polymerase chain reaction (RT-PCR) method. After 6 and 21 days of incubation an initiation of mineralization and the presence of newly formed bone at the surface of the composites were shown after evaluation of ALP activity, DNA content, SEM and histological staining. Expression of bone-specific genes confirmed the bone-like character of these composites and different effects of PLGA or HA granulae on the osteogenic differentiation of human MCLPCs in vitro. The results of this study support the concept that substrate signals significantly influence MCLPCs growth, differentiation, mineralization and gene expression in vitro, and that the use of these cells in the manufacturing of 3-D cell/HA composites is a promising approach for load bearing bone reconstruction in the maxillofacial region in vivo.

Purpose: The purpose of this study was to analyze the interaction between osteoblast-like cells isolated from mandibular bone and hydroxyapatite ceramic bone substitute obtained from calcified red algae to assess the growth and differentiation of adherent cells on this biomaterial. Materials and Methods: The macroporous ceramic material C GRAFT/Algipore (The Clinician’s Preference LLC, Golden, CO) is composed of 100% hydroxyapatite and possesses specific mechanical and physiochemical properties. Osteoblast-like cells were seeded on 200 mg of biomaterial and cultured for 6 and 21 days under osteogenic differentiation conditions. Specific alkaline phosphatase activity, DNA, and protein content of the proliferating cells were analyzed. The morphology of the cells in contact with the biomaterial was examined by scanning electron microscopy. The osteoblastic phenotype of the cells was confirmed by analysis of the expression of bone-specific genes (osteocalcin, osteopontin and collagen type I) by semi-quantitative reverse transcriptase polymerase chain reaction. Results: The DNA and protein content increased over the culture period. Scanning electron microscopy showed cells spreading on the surface of the biomaterials, covering the macropores, and colonizing the depth of the particles. The analysis of the expression patterns of bone-related genes confirmed the osteoblastic phenotype of the cultured cells. Conclusion: The results of this study showed that hydroxyapatite ceramic bone substitute obtained from calcified red algae support the proliferation and differentiation of human osteoblast-like cells on its surface in vitro and might be suitable for use as scaffolds in tissue engineering strategies in vivo.

Purpose Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering. Materials and methods Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician’s Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor α1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis. Results Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period. Conclusion We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.