Surface- and nonsurface-dependent in vitro effects of bone substitutes on cell viability

The aim of the present in vitro study was to evaluate the influence of different bone substitute materials (BSM) on the viability of human primary osteoblasts (PO), bone marrow mesenchymal cells (BMMC), and nonadherent myelomonocytic cells (U937). Six different bone substitute materials were tested: Bio-Oss Spongiosa (BOS), Tutodent Chips (TC), PepGen P-15 (P-15), Ostim (OM), BioBase (BB), and Cerasorb (CER). Cells were cultivated on comparable volumes of BSM in 96-well plates. Cell culture-treated polystyrol (Nunclon Delta surface; C) served as positive control. After 2 h and 3, 6, 10, and 14 days, viability of cells was evaluated using a standardized ATP viability assay (CellTiter Glo). Nonsurface-dependent effects of the materials were separately tested using nonadherent U937 suspension cells. For statistical analysis, the Mann-Whitney test was used. Results were considered statistically significant at P < 0.05. Cell viability of PO increased significantly on TC, C, and CER followed by BB. No changes were found for P-15 and decreasing viability for BOS and OM. BMMC showed similar results on C, TC, CER, and P-15. Lower viability for BB and no viability could be detected for BOS and OM (Mann-Whitney test, respectively). Nonadherent cells displayed increasing viability in presence of CER, BB, and BOS. No changes were observed for TC and P-15, whereas for OM, no viability was detected after a maximum cultivation period of 3 days. It was concluded that granular hydroxyapatite (HA; TC, BOS, P-15) and alpha- and beta-tricalciumphosphate (CER, BB) support, whereas nanosized HA (OM) limit or even inhibit surface- and nonsurface-related cell viability in the in vitro model used.     

In vitro inhibitory effects of Polygonum cuspidatum on bacterial viability and virulence factors of Streptococcus mutans and Streptococcus sobrinus

OBJECTIVES: Polygonum cuspidatum has been used in Korean folk medicine to improve oral hygiene. This study was performed to evaluate the effects of methanol extract from root of P. cuspidatum (MEP) on bacterial viability and the virulence factors of Streptococcus mutans and Streptococcus sobrinus. METHODS: To test the effects of MEP on bacterial viability, we determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 20 bacterial strains, including S. mutans and S. sobrinus, using a micro-dilution assay. In case of S. mutans and S. sobrinus, the assays for time-kill and bacterial growth rate at sub-MIC concentrations were also performed. To determine effects of the extract on the virulence factors of S. mutans and S. sobrinus, the assays for sucrose-dependent adherence, water-insoluble glucan formation, glycolytic acid production, and acid tolerance were performed at sub-MIC levels. Phytochemical analysis for constituents of MEP was carried out. RESULTS: MEP showed a broad antibacterial range (MIC 0.5-4 mg/ml). The MBC was two to four times higher than the MIC. The time-kill curves showed S. mutans and S. sobrinus were significantly killed after 1h of incubation. At sub-MIC levels, doubling times of S. mutans and S. sobrinus dose-dependently increased up to 211% and 123%, respectively. At sub-MIC levels, MEP also showed inhibitory effects on the virulence factors of S. mutans and S. sobrinus in a dose-dependent fashion. Phytochemical analysis revealed the presence of alkaloids, sterol/terpenes, tannins, flavonoids, and carbohydrates. CONCLUSION: These data indicate that MEP has inhibitory effects on bacterial viability at higher concentrations (> or =MIC) and the virulence factors of S. mutans and S. sobrinus at sub-MIC concentrations, suggesting that it might be useful for the control of dental plaque formation and subsequent dental caries formation.     

In vitro viability, mitogenicity and clonogenic capacities of periodontal ligament fibroblasts after storage in four media supplemented with growth factors

Abstract – The choice of storage medium for preserving traumatically avulsed teeth is important for the success of future replantation. The objective of this study was to evaluate the effectiveness of growth factors (IGF‐1 and PDGF‐BB) when added to storage media in preserving the functional abilities of cultured periodontal ligament fibroblasts (PDLF). The evaluated storage media were: ViaSpan, Hanks’ balanced salt solution (HBSS), α minimal essential medium (α MEM), and α MEM supplemented with FCS and antibiotic (α MEM‐S). PDLF were obtained from explants of human healthy extracted teeth. Plates with confluent PDLF were soaked in the various media supplemented with IGF‐1 (10 ng/ml) and PDGF‐BB (4 ng/ml) for 2, 8 and 24 h at room temperature (24 °C). The control group was incubated with the examined storage media without growth factors at 24 °C. An additional control group was incubated with culture medium at 37 °C without growth factors. After incubation, the viability of the cells was determined by Trypan blue exclusion test. Viable cells were then analyzed for mitogenic (with thymidine) and clonogenic (by culturing one cell/well) capacities. Storage of PDLF with growth factors (GF) for 2, 8 and 24 h decreased their vitality by only 3% (not statistically significant). The mitogenicity of PDLF stored for 2, 8 and 24 h in various media with GF was statistically comparable to that of the control group. Generally, the highest mitogenic capacity of PDLF stored with or without GF was found after 8 h of storage. Increasing the storage period to 24 h decreased the mitogenic capacity of the cells stored with GF by only 10–40% compared to the control group. In contrast, the clonogenic capacity of PDLF stored with GF increased with increasing storage periods by 100–300%, and the highest clonogenic capacity was found in most storage media after 24 h of storage with GF. The highest clonogenic and mitogenic capacities were found in cells stored in HBSS followed by α MEM‐S. The mitogenic and clonogenic capacities of PDLF stored in various media supplemented with GF for 2–8 h were generally lower than without GF supplementation. The mitogenic and clonogenic effects of GF‐supplementation was observed only after 24 h of storage. After 24 h of storage with GF, the clonogenic capacity increased by 8–224% and the mitogenicity by 20–37%, except in cells stored in α MEM (‐1%). However, these differences were generally not statistically significant. In conclusion, the mitogenic and clonogenic effects of GF were observed only after 24 h of storage at room temperature. HBSS and α MEM‐S supplemented with GF were the most effective media for preserving the viability, mitogenicity and clonogenic capacity of PDLF stored for 24 h at room temperature. For short periods of storage (2 and 8 h), HBSS and α MEM‐S without GF were preferable.     

In vitro effects of enamel matrix proteins on rat bone marrow cells and gingival fibroblasts

Emdogain (EMD), a formulation of Enamel Matrix Proteins (EMP), is used clinically for periodontal regeneration, where it stimulates cementum formation and promotes gingival healing. In this study, we investigated the in vitro effects of EMD on rat bone marrow stromal cells (BMSC) and gingival fibroblasts (GF). EMD (at 25 micro g/mL) increased the osteogenic capacity of bone marrow, as evidenced by approximately three-fold increase in BMSC cell number and approximately two-fold increase in alkaline phosphatase (ALP) activity and mineralized nodule formation. The presence of EMD in the initial stages (first 48 hrs) of the culture was crucial for this effect. In contrast, EMD did not induce osteoblastic differentiation of GF (evidenced by lack of mineralization or ALP activity) but increased up to two-fold both their number and the amount of matrix produced. These in vitro data on BMSC and GF could explain the promotive effect of EMD on bone formation and connective tissue regeneration, respectively.     

降钙素对人骨巨细胞瘤组织中破骨细胞的影响

目的观察降钙素对人骨巨细胞瘤(giant cell tumor of bone,GCT)组织中纯化的破骨细胞的数量以及细胞核因子-κB受体活化因子配基(RANKL)蛋白表达的影响。方法利用破骨细胞贴壁快以及耐胰蛋白酶的特性,采用0.25%胰蛋白酶和0.2%I型胶原酶来分离纯化骨巨细胞瘤中的破骨细胞,设立对照组和试验组,在实验组培养液中加入浓度为10-8mol/L的降钙素,以抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞数目、形态,用免疫组化方法观察RANKL的表达。结果从骨巨细胞瘤中纯化方法得到的破骨细胞数量较多,加药组破骨细胞数目较对照组明显减少(P<0.05)。RANKL的蛋白表达两组未见明显差异(P>0.05)。结论降钙素可显著的抑制骨巨细胞瘤中纯化破骨细胞的分化和功能。     

In vitro comparison of new bisphosphonic acids and zoledronate effects on human gingival fibroblasts viability,inflammation and matrix turnover

Objectives

Bisphosphonates (BPs) are drugs clinically used in resorptive diseases. It was already proved that some clinically relevant BPs can inhibit a class of enzymes called matrix metalloproteinases (MMPs), required during tissue remodelling. Combining the arylsulfonamide function with the bisphosphonic group, several compounds were synthesized to obtain selective inhibitors of MMPs. The aim of the present study was to compare the effect of zoledronic acid (ZA), the most potent bisphosphonate available as therapy, with new sulfonamide containing BPs in an in vitro model of human gingival fibroblasts (HGFs).

Materials and methods

Western blot was used to measure procollagen I, β1 integrin MMP-8 and MMP-9, phase contrast and MTT for cell viability; L-lactate-dehydrogenase (LDH) measurement was performed for toxicity evaluation and ELISA for prostaglandin E₂ (PGE₂) secretion assessment.

Results

When compared with ZA, the treatment with the newly synthesized compounds shows increasing viability, procollagen I expression and decreased expression of β1 integrin in HGFs. Higher levels of released LDH, PGE₂ and MMP-9 expression are recorded in ZA-treated HGFs. Increased levels of MMP-8 are recorded in newly synthesized compounds-treated samples.

Conclusions

These findings allowed to conclude that new tested BPs did not affect HGFs viability and adhesion, did not induce cellular toxicity, were not responsible for inflammatory event induction and could preserve the physiological matrix turnover.

Clinical Relevance

It could be hypothesized that the new molecules were better tolerated by soft tissues, resulting in lesser side effects.

     

Activation of human platelet-rich plasmas: effect on growth factors release, cell division and in vivo bone formation

OBJECTIVES: Aims of this controlled study were to determine the effects of activated human platelet-rich plasmas (PRPs) on early and mature bone formation in vivo, and to characterize the effect of PRP activation on growth factors release and endothelial cell division in vitro. MATERIAL AND METHODS: PRPs were prepared from four volunteers with the platelet concentrate collector system (PCCS) system and activated with three concentrations of calcium and thrombin. Platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and interleukin-1beta (IL-1beta) levels released in supernatants were measured by ELISA, at time 0, 1h, 24h and 6 days following PRP activation. Mitogenic potential of PRP supernatants were tested on endothelial cells in vitro, and the effects of activated human PRPs on bone formation in vivo were measured in athymic rats by micro-CT analyses. RESULTS: Activation of PRPs with calcium and thrombin triggered an immediate release of VEGF, PDGF-BB and TGF-beta and a delayed release of IL-1beta in PRP supernatants. Higher endothelial cell division was observed with supernatants from activated PRPs than from non-activated PRPs. Positive correlations were observed between VEGF levels and endothelial cell division and bone formation. A negative correlation was also found between PDGF-BB concentration and bone formation. However, early and mature bone formations with activated PRPs did not significantly differ from the ones obtained in the control group. CONCLUSIONS: Activation of PRPs with calcium and thrombin regulates growth factors release and endothelial cell division in vitro. However, activated PRPs does not improve the early or mature bone formations in vivo in this athymic rat model.     

牙源性囊肿和成釉细胞瘤体外骨吸收的实验研究

目的定量分析牙源性角化囊肿和成釉细胞瘤的体外骨吸收效应,探讨其颌骨吸收机制。方法收集25例牙源性囊肿[牙源性角化囊肿(OKC)14例、牙源性角化囊肿伴感染6例、含牙囊肿(DC)5例]和7例成釉细胞瘤的新鲜组织块行体外培养(24h),取其上清液与SD大鼠(新生5天)颅盖骨培养体系继续培养48h,以原子分光光度计法检测培养体系上清液中的Ca2+含量,从而判断不同牙源性病损在体外导致骨吸收作用的差异。同时采用放射免疫技术检测牙源性病损体外培养上清液中的骨吸收相关因子:白细胞介素6(IL6)、肿瘤坏死因子α(TNFα)、前列腺素E2(PGE2)、骨钙素(BGP)和降钙素(CT)等的含量。结果各组牙源性囊肿和肿瘤引起大鼠颅盖骨培养Ca2+析出的浓度显著高于空白组(P<0.01);OKC伴感染组Ca2+浓度显著高于OKC组和成釉细胞瘤组(P<0.05)。各组牙源性囊肿和成釉细胞瘤培养上清液中IL6、TNFα、PGE2和CT含量显著高于空白对照组(P<0.05);OKC组和OKC伴感染组IL6含量显著高于成釉细胞瘤组(P<0.05);OKC伴感染组CT含量显著高于OKC组和含牙囊肿组(P<0.05)。这些因子和Ca2+含量的相关性分析结果显示,IL6与钙值之间呈显著性正相关(P<0.01)。结论颌骨牙源性病损在体外可促进骨吸收,此作用可能与其产生的某些细胞因子有关。     

In vitro effect of microwave irradiation on the retentive force of magnets

STATEMENT OF PROBLEM: Few studies have addressed the possible effect(s) of microwave irradiation on the magnetic properties of "permanent" magnets during the fabrication of dental and maxillofacial prostheses. PURPOSE: The objective of this study was to investigate the influence of microwave irradiation energy on the retentive force of a magnetic attachment system used in maxillofacial prosthetic rehabilitation. MATERIAL AND METHODS: A heat-polymerized PMMA disk (6 cm in diameter) was fabricated. Seven magnets were placed around the wafer in a circumferential fashion: 1 in the center and 6 surrounding it. The 7 magnets were spaced 2 cm from their respective centers. Seven heat-polymerized PMMA cylinders were also used, and a magnet (counter-magnet) was placed in the center of each even with the cylinder's surface. Once the investment had set (45 minutes after mixing), the 7 counter-magnets in the cylinders were placed against the 7 magnets in the acrylic wafer. A second mixture of investment material was added. The flasks were separated, and the acrylic wafer was removed to accommodate the 7 counter-magnets in the base mold in the same geometric configuration and to serve as a "spacer" for the silicone material. A 1:1 mixture of medical grade elastomer (MDX4-4210) and medical adhesive silicone (type A) was packed and compressed, the molds were reclamped, and the excess silicone was removed. The elastomer/silicone wafer was packed and compressed into the test and base molds, the molds were reclamped, and excess silicone was removed. The first group of magnets, designated Group A, received microwave irradiation for 5 minutes at low power (112 W). This procedure was repeated for each group of magnets at the following polymerizing times (n=14): Group B, 10 minutes; Group C, 15 minutes; Group D, 20 minutes; Group E, 25 minutes; Group F, 30 minutes; and Group G, 35 minutes. Measurements of retentive force (N) at 10 mm/min ramp rate of speed of separation was conducted. The specimen rate read 5.0 points/second. Data were analyzed using a 1-way analysis of variance (alpha=.05); individual mean values were compared using the Tukey test (alpha=.05). RESULTS: There were no statistical differences in retentive force between groups D, E, and F (20, 25, and 30 minutes, respectively) or between groups A, B, C, D, F, and G (5, 10, 15, 20, 30, and 35 minutes, respectively). When the microwave-irradiated groups A through G were compared with the control group, there was a significant difference (P<.05) in retentive force (N). Group E (25 minutes) showed the largest reduction of retentive force (0.3 N, a reduction of 12%). CONCLUSION: If a prosthesis is processed using a microwave and contains samarium cobalt magnets, the retentive force may be reduced up to 12% under specific conditions.     

In vitro approach to evaluate potential harmful effects of beer on teeth

OBJECTIVE: The purpose of the present work was to examine some properties of different brands of beer manufactured in Brazil that may be important to oral health. METHODS: Samples from seven different beer brands were analyzed for pH, titratable acidity, calcium and phosphate concentrations. Demineralization experiments were carried out by incubating samples with crown tooth particles (40-80 mesh) at 37 degrees C under agitation (100 strokes/min). RESULTS: The pH was lower than 4.0 for three of the seven samples and higher than 4.0 for the others. The amount of titratable acidity, expressed as the volume of 0.1N NaOH solution consumed to raise the initial pH to 7.0, and the concentrations of calcium and phosphate varied. Calcium concentration ranged from 0.21 to 1.59 micromol/ml, while phosphate concentration varied from 0.048 to 0.094 micromol/ml. Calcium released to the incubation medium was proportional to the time of incubation up to 5min. Maltose, a disaccharide, was detected in all samples studied. CONCLUSION: Differences in the properties examined indicated that some brands of beer studied may have potential dental effects.     

淫羊藿对口腔各矿化组织破骨细胞性骨吸收的体外实验研究

目的 探讨中药淫羊藿抑制破骨细胞性骨吸收的作用。方法 体外分离、培养兔破骨细胞，与玻片及灭活牙片共同培养，加入不同浓度淫羊藿注射液。HE、原位末端标记染色玻片上的破骨细胞，观察其形态结构的改变，并观察破骨细胞在牙片上形成的吸收陷窝数目及面积的变化。结果 HE染色可见用药组破骨细胞胞质浓缩，核固缩深染，部分细胞出现核分裂。原位末端标记结果显示，用药组破骨细胞胞质皱缩，胞核呈棕褐色，胞质淡杂。提示淫羊藿可诱导破骨细胞凋亡，抑制骨吸收。用药组与非用药组破骨细胞凋亡率差异有显著性，吸收陷窝数目、面积差异也有显著性，随浓度增加抑制作用增强。结论 淫羊藿可诱导破骨细胞凋亡，抑制骨吸收，并随浓度增加抑制作用增强。     

In vitro cell death induced by irradiation and chemicals relevant for dental applications; dose-response and potentiation effects

Resin-based dental materials polymerized using blue light are frequently used in dental practice and may come in contact with the oral mucosa. Remnants from oral hygiene product ingredients, such as sodium lauryl sulfate (SLS), add to the chemical exposure of the mucosa. The aim of the present in vitro study was to elucidate the cytotoxic effects in terms of apoptosis and necrosis after exposures to combinations of an adhesive (0.5% and 0.6%), SLS (concentration range 0.0025%-0.0075%), and irradiation from a dental curing lamp (radiant exposure of 8 J cm(-2)). The test system chosen was rat submandibular salivary gland acinar cells, and the cytotoxic effects were measured by fluorescence microscopy and flow cytometry methods. Cytotoxicity was observed as a result of irradiation. The most pronounced cytotoxic effects were seen in cells exposed to a combination of adhesive and SLS compared with those exposed to either agent alone. Necrosis was the dominating form of cell death for all exposures, except for the highest concentration of SLS. Apoptosis was dose-dependent on SLS in the rat submandibular acinar cells. Cytotoxic considerations of dental materials should include contributions from irradiation and other chemicals that might be present in the oral cavity.     

The effects of particulate metals on cell viability of osteoblast-like cells in vitro

Effects of fifteen particulate objects, fourteen metals and one non-metal on cell viability of osteoblast-like cells were studied in vitro, to determine whether an adverse effect on cells could be induced by the particulate form or soluble ions. The Al, Ti, Zr, Nb, Ta, Cr, Mo, and Fe particulates depressed cell viability at higher particulate concentrations, but their extracts yielded no effect on cells except for Mo. On the other hand, little difference in cell viability between particulates and extracts was observed for Cu, Si, V, W, and Co. However, Mn and Ni yielded more adverse effects on cells in the case of the particulates than the extracts. These findings suggested that the effects of particulates on cells depended upon the direct effects of contact between particulates and cells, the indirect effects of dissolved ions and the kinds of particulate elements.     

Local simvastatin effects on mandibular bone growth and inflammation

BACKGROUND: Simvastatin has been shown to increase bone growth when applied topically to murine bone; however, it causes considerable soft tissue inflammation at high doses (2.2 mg), making future clinical use problematic. This study evaluated the effect of lower simvastatin doses and cyclooxygenase (COX) synthase inhibitors on tissue inflammation and bone growth in rats and gene expression in mice. METHODS: Adult female rats were untreated or treated with a single dose of 0.1, 0.5, 1.0, 1.5, or 2.2 mg simvastatin in methylcellulose gel in a polylactic acid membrane (SIM) on the lateral aspect of the mandible. The contralateral mandible side was implanted with methylcellulose gel/polylactic acid membrane alone (GEL), and five rats in each dose pairing were evaluated histomorphometrically after 3, 7, and 24 days. Subsequent rats were similarly treated with 0.5 mg simvastatin (optimal dose) and daily intraperitoneal injections of COX-2 inhibitor (NS-398; 1 mg/kg x 7 days; N = 16), general COX inhibitor (indomethacin; 1 mg/kg x 7 days; N = 16), or no inhibitor (N = 10) and evaluated histomorphometrically after 7 or 24 days by analysis of variance (ANOVA). Gene arrays were also used to evaluate osteogenic gene expression from 0.5 mg simvastatin in murine calvaria (N = 12). RESULTS: There was a 45% increase in bone area with 0.5 mg simvastatin versus gel control (P <0.001; similar to the 2.2-mg dose), and clinical swelling was reduced compared to the high simvastatin dose (P <0.05). The 0.1-mg simvastatin dose failed to stimulate significant bone growth. NS-398 and indomethacin reduced inflammation and bone growth. Simvastatin significantly upregulated procollagen, fibronectin, and matrix metalloproteinase-13 genes. CONCLUSION: Reducing the simvastatin dose from 2.2 to 0.5 mg reduced inflammation to a more clinically acceptable level without sacrificing bone-growth potential, but COX-associated inflammation appears to be necessary for in vivo bone growth.