Bioactivity of endodontic biomaterials on dental pulp stem cells through dentin

OBJECTIVES@#This study investigated the indirect effect of calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA), as 2 calcium silicate-based hydraulic cements, on human dental pulp stem cells (hDPSCs) through different dentin thicknesses.@*MATERIALS AND METHODS@#Two-chamber setups were designed to simulate indirect pulp capping (IPC). Human molars were sectioned to obtain 0.1-, 0.3-, and 0.5-mm-thick dentin discs, which were placed between the 2 chambers to simulate an IPC procedure. Then, MTA and CEM were applied on one side of the discs, while hDPSCs were cultured on the other side. After 2 weeks of incubation, the cells were removed, and cell proliferation, morphology, and attachment to the discs were evaluated under scanning electron microscopy (SEM). Energy-dispersive X-ray (EDXA) spectroscopy was performed for elemental analysis. Alkaline phosphatase (ALP) activity was assessed quantitatively. The data were analyzed using the Kruskal-Wallis and Mann-Whitney tests.@*RESULTS@#SEM micrographs revealed elongated cells, collagen fibers, and calcified nucleations in all samples. EDXA verified that the calcified nucleations consisted of calcium phosphate. The largest calcifications were seen in the 0.1-mm-thick dentin subgroups. There was no significant difference in ALP activity across the CEM subgroups; however, ALP activity was significantly lower in the 0.1-mm-thick dentin subgroup than in the other MTA subgroups (p < 0.05).@*CONCLUSIONS@#The employed capping biomaterials exerted biological activity on hDPSCs, as shown by cell proliferation, morphology, and attachment and calcific precipitations, through 0.1- to 0.5-mm-thick layers of dentin. In IPC, the bioactivity of these endodontic biomaterials is probably beneficial.

Bioactivity of endodontic biomaterials on dental pulp stem cells through dentin

OBJECTIVES: This study investigated the indirect effect of calcium-enriched mixture (CEM) cement and mineral trioxide aggregate (MTA), as 2 calcium silicate-based hydraulic cements, on human dental pulp stem cells (hDPSCs) through different dentin thicknesses.MATERIALS AND METHODS: Two-chamber setups were designed to simulate indirect pulp capping (IPC). Human molars were sectioned to obtain 0.1-, 0.3-, and 0.5-mm-thick dentin discs, which were placed between the 2 chambers to simulate an IPC procedure. Then, MTA and CEM were applied on one side of the discs, while hDPSCs were cultured on the other side. After 2 weeks of incubation, the cells were removed, and cell proliferation, morphology, and attachment to the discs were evaluated under scanning electron microscopy (SEM). Energy-dispersive X-ray (EDXA) spectroscopy was performed for elemental analysis. Alkaline phosphatase (ALP) activity was assessed quantitatively. The data were analyzed using the Kruskal-Wallis and Mann-Whitney tests.RESULTS: SEM micrographs revealed elongated cells, collagen fibers, and calcified nucleations in all samples. EDXA verified that the calcified nucleations consisted of calcium phosphate. The largest calcifications were seen in the 0.1-mm-thick dentin subgroups. There was no significant difference in ALP activity across the CEM subgroups; however, ALP activity was significantly lower in the 0.1-mm-thick dentin subgroup than in the other MTA subgroups (p < 0.05).CONCLUSIONS: The employed capping biomaterials exerted biological activity on hDPSCs, as shown by cell proliferation, morphology, and attachment and calcific precipitations, through 0.1- to 0.5-mm-thick layers of dentin. In IPC, the bioactivity of these endodontic biomaterials is probably beneficial.

Exposure of neonatal mice to Sevoflurane may induce male germ cell apoptosis in testicular tissue after puberty

Background: common use of sevoflurane in congenital defects during repeated surgeries may have detrimental effects on spermatogenesis after puberty

Objective: this study investigated sevoflurane effects on spermatogenesis process in male mature mice after exposure in prepubertal time

Materials and Methods: 24 neonatal NMRI male mice were randomly classified in three groups. Experimental 1 and 2 groups [exposure to 1 minimum alveolar concentration [MAC] and 2 MAC sevoflurane, respectively in 2 lit/min oxygen [O[2]] for 7 days [30 min, daily] and control. All groups were sacrificed after 2 months. Histological assessment, immunohistochemistry and apoptosis process was done. Bax and Bcl2 expression was evaluated in the testicular tissue by real time Poly Chain Reaction

Results: our results showed that the integrity of testicular tissue was preserved in both experimental groups. Count of spermatogonial cells had significant decrease in group 2 compared to others. The rate of apoptosis in spermatogonial cells was 15+/-3% and 9+/-2% in the group 2 and 1, respectively. Also, Bax/Bcl[2] ratio was 0.2615, 1.0070 and 9.3657 in control, experimental group 1 and 2, respectively. This result was significant [p

Conclusion: continuous exposure of 2 MAC sevoflurane in 2 lit/min O[2] simultaneous during prepubertal may create more testicular tissue damage in terms of cellular and molecular function compared to continuous exposure to lower level of sevoflurane by increase in ratio of Bax/Bcl[2] and apoptosis in germ cells after puberty

Application of stereological methods for unbiased estimation of sperm morphology in the mice induced by busulfan / 대한해부학회지

Busulfan is an anticancer drug, which causes the apoptosis germ cells and azoospermia in humans and animals. Abnormal morphology of spermatozoa related to the male infertility. The sperm morphology is evaluation of sperm size, shape and appearance characteristics should be assessed by carefully observing a stained sperm sample under the microscope. Evaluation of sperm morphology has been considered as one of the most important factors for a successful fertilization and determining sperm quality. The mice were assigned to tow experimental groups: control and busulfan. Each group included six mice that were housed under standard conditions. The volume was estimated using the nucleator method. The sperm's flegellum and mid-piece length was estimated by counting the number of intersections between the tails and Merz grid test line in an unbiased counting frame, superimposed on live images of sperms. Our results demonstrated a significant different in the volume and surface of the sperm's head and the length of the sperm's flagellum in the control and busulfan groups. Busulfan can effect on the volume of the sperm's head and the length of the sperm's flagellum in rat.

Expression of glycogen synthase kinase 3-beta [GSK3-beta] gene in azoospermic men

The Wnt/beta- The Wnt/beta-catenin signaling pathway is involved in many developmental processes in both fetal and adult life; its abnormalities can lead to disorders including several types of cancers and malfunction of specific cells and tissues in both animals and humans. Its role in reproductive processes has been proven. This study was designed to evaluate the expression of the key regulator of this signaling pathway GSK3-beta and its presumed role in azoospermia. WNT3[a] protein concentration and GSK3-beta gene expression levels were measured and compared between two groups of infertile men. The test groups consisted of 10 patients with obstructive and 10 non-obstructive azoospermia. The control group was selected among healthy men after vasectomies that were willing to conceive a child using a testicular biopsy technique. Samples were obtained by testicular biopsy and screened for the most common mutations [84, 86 and 255] in the SRY region before analyzing. GSK3-beta gene expression was assessed quantitatively by real time-PCR. The WNT3[a] protein concentration had no significant difference between the two test groups and controls. Expression of GSK3-beta was down-regulated in non-obstructive azoospermia [3.10 +/- 0.19] compared with normal [7.12 +/- 0.39] and obstructive azoospermia [6.32 +/- 0.42] groups [p=0.001]. Down-regulation of GSK-3beta may cause to non-obstructive azoospermia. Regulation and modification of GSK-3beta gene expression by drugs could be used as a therapeutic solution

[Alkaline phosphatase activity level of osteoblast cells on three-dimensional chitosan-gelatin/hydroxyapatite composite scaffolds]

In recent years, composite scaffolds made of polymers and bioactive ceramics have found numerous applications in bone tissue engineering due to their superior properties. Among various polymers, chitosan [Cs] and gelatin [Gel] gained more attention because of their desirable properties. In this study, by using these two polymers and hydroxyapatite [HA] which resembles inorganic phase of human bone, three-dimensional composite scaffolds were prepared by freeze-drying method and behavior of osteoblast cells on them was evaluated. This study aimed at preparing an appropriate bioactive scaffold for bone tissue engineering. In order to evaluate the effect of gelatin concentration on biological properties of scaffolds, three different concentrations of gelatin [0, 10, and 20%] were added to composite scaffolds. For assessment of the biological properties of scaffolds, osteoblast cells were cultured on the composite scaffolds and their behavior was monitored. Scanning electron microscopy [SEM] analysis and alkaline phosphatase activity [ALP] test were carried out to assess the mentioned factors. SEM analysis showed high cell attachment and proliferation on the scaffolds. Alkaline phosphatase activity of osteoblast cells indicated the suitability of composite scaffolds containing a higher concentration of gelatin compared to other concentrations as well as overall high activity of osteoblast cells on all scaffolds. Cs/Gel/HA bioactive composite scaffold is recommended for bone tissue regeneration purposes as a suitable scaffold

Isolation and in vitro characterization of mesenchymal stem cells derived from the pulp tissue of human third molar tooth

It is still controversial that the stem cells isolated from human dental pulp meets the criteria for mesenchymal stem cells [MSCs]. The aim of the present study was to examine whether or not they are MSCs, or are distinct stem cells population residing in tooth pulp. Adherent fibroblastic cells in the culture of pulp tissue from human third molars were propagated through several successive subcultures. Passaged-3 cells with a tendency to differentiate into odontoblastic cells were used to examine the key properties of MSCs including typical tripotent differentiation potential into bone, cartilage and adipose cell lineages and the expression of typical surface antigens. Moreover, they were examined for growth capacity in culture. Dental pulp stem cells successfully progressed towards differentiation among three skeletal cell lineages. More than 90% of the cell population exhibited the expression of surface antigens known to be found on mesenchymal lineages such as CD105, CD90, CD44, and CD73, while only less than 2% expressed endothelial-hematopoietic epitopes including CD56, CDllb, CD34, CD31, CD33, and CD45. The cells exhibited a relatively high proliferation capacity with population doubling time of about 21.9 hours. The dental pulp stem cells are of MSC population, and may be considered suitable for use in regenerative medicine, owing to their relatively rapid rate of in vitro propagation

Ex vivo expansion and differentiation of mesenchymal stem cells from goat bone marrow

Mesenchymal stem cells [MSCs] from large animals as goat which is genetically more closely related to human have rarely been gained attentions. The present study tried to isolate and characterize MSCs from goat bone marrow. Fibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures. Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineages to determine their MSC nature. Differentiations were determined by RT-PCR analysis of related gene expression. To identify the best culture conditions for propagation, passage-3 cells were plated either at varying cell densities or different fetal bovine serum [FBS] concentrations for a week, at the end of which the cultures were statistically compared with respect to the cell proliferation. In this study, we also determined goat MSC population doubling time [PDT] as the index of their in vitro expansion rate. Passage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in both specific staining as well as the specific gene expression profile. Moreover, there appeared to be more expansion when the cultures were initiated at 100 cells/cm[2] in a medium supplemented with 15% FBS. A relatively short PDT [24.94 +/- 2.67 hr] was a reflection of the goat MSC rapid rate of expansion. Taken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletal cell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBS concentration

Mesenchymal stem cell isolation from the removed medium of rat's bone marrow primary culture and their differentiation into skeletal cell lineages

In all protocols for isolation of mesenchymal stem cells [MSCs], a few days after culture initiation, the medium were discarded along with its contents of non-adherent cells and the adherent cell population kept and expanded as MSCs population. In the present study, attempt was made to expand the cells suspended in removed medium of primary culture and compare them with the adherent cell population. Four days after rat's bone marrow culture initiation, medium of the culture was collected and its suspended cells were culture-expanded in parallel with adherent cells till passage 3. During the culture period, the cells from either group were statistically compared with respect of the time required for cell confluency [the stage in which cells cover the entire surfaces] as an index of growth rate. At the end, the cells from both cultures were evaluated in terms of their differentiation potential. The primary culture of the cells from removed medium contained large colonies of spindle-shaped cells that reached into confluency after 5.36 +/- 0.5 days, while those from the adherent population possessed small colonies reaching into confluency in 8.09 +/- 0.70 days. According to the results, at all studied passages, the cells of removed medium were significantly [p<0.05] achieved confluency in shorter time than the adherent population. Moreover, the cells from either culture could easily differentiate into bone, cartilage and adipose cells. It seems that some cells from removed medium, usually discarded in medium substitution, are MSCs possessing more growth rate than the primarily adherent cell population