Stem Cells from Human Exfoliated Deciduous Teeth Modulate Early Astrocyte Response after Spinal Cord Contusion.

The transplantation of stem cells from human exfoliated deciduous teeth (SHED) has been studied as a possible treatment strategy for spinal cord injuries (SCIs) due to its potential for promoting tissue protection and functional recovery. The aim of the present study was to investigate the effects of the early transplantation of SHED on glial scar formation and astrocytic reaction after an experimental model of SCI. Wistar rats were spinalized using the NYU Impactor. Animals were randomly distributed into three groups: control (naive) (animal with no manipulation); SCI (receiving laminectomy followed by SCI and treated with vehicle), and SHED (SCI rat treated with intraspinal SHED transplantation, 1 h after SCI). In vitro investigation demonstrated that SHED were able to express mesenchymal stem cells, vimentin and S100B markers, related with neural progenitor and glial cells, respectively. The acute SHED transplantation promoted functional recovery, measured as from the first week after spinal cord contusion by Basso, Beattie, and Bresnahan scale. Twenty-four and 48 h after lesion, flow cytometry revealed a spinal cord vimentin+ cells increment in the SHED group. The increase of vimentin+ cells was confirmed by immunofluorescence. Moreover, the bioavailability of astrocytic proteins such as S100B and Kir4.1 shown to be increased in the spinal cord of SHED group, whereas there was a glial scar reduction, as indicated by ELISA and Western blot techniques. The presented results support that SHED act as a neuroprotector agent after transplantation, probably through paracrine signaling to reduce glial scar formation, inducing tissue plasticity and functional recovery.

Laser treatment contributes to maintain membrane integrity in stem cells from human exfoliated deciduous teeth (shed) under nutritional deficit.

This study aimed to analyze the effects of laser irradiation on the membrane integrity and viability of stem cells from human exfoliated deciduous teeth (SHED) that were kept in serum starvation. Nutritional deficit was used to mimic the cellular stress conditions of SHED isolation for regenerative dental approaches, where laser therapy could be beneficial. SHED were cultured under serum starvation (MEMα + 1%FBS) for 1 or 24 h pre-irradiation (protocols A and B, respectively). Then, cells received low-level laser therapy (LLLT; 660 nm) at 2.5 J/cm2 (0.10 W; groups I and V), 5.0 J/cm2 (0.20 W; groups II and VI), 7.5 J/cm2 (0.30 W; groups III and VII), or remained non-irradiated (groups IV and VIII). During irradiation, cells were maintained in 1% FBS (groups I-IV) or 10% FBS (normal culture conditions; groups V-VIII). Membrane integrity was evaluated by quantifying lactate dehydrogenase (LDH) release (immediately after irradiation), and cell viability was assessed by the MTT assay (24, 48, and 72 h post-irradiation). Serum starvation did not alter LDH release by non-irradiated SHED, while LDH release decreased significantly in groups irradiated in 1% FBS (I and III), but not in groups irradiated in 10% FBS (V-VII), regardless the pre-irradiation conditions (protocols A/B). Cell viability was significantly higher 24 h after irradiation, in most protocol A groups. In contrast, cell viability remained mostly unaltered in protocol B groups. LLLT contributed to maintain membrane integrity in SHED subjected to nutritional deficit before and during irradiation with 0.10 or 0.30 W. Short serum starvation before irradiation improved SHED viability at 24 h post-irradiation.

Short-term evaluation of photobiomodulation therapy on the proliferation and undifferentiated status of dental pulp stem cells.

The aim of this in vitro study was to analyze the effect of photobiomodulation therapy (PBMT) on the proliferation and undifferentiating status of stem cell from human exfoliated deciduous teeth (SHEDs). PBMT was carried out with an aluminum gallium indium phosphide (InGaAlP) diode laser in contact and punctual mode (continuous wave, 660 nm, 20 mW, 0.028 cm2, and average energy densities of 1 (1 s), 3 (4 s), 5 (7 s), 10 (14 s), 15 (21 s), or 20 (28 s) J/cm2 per point). The immunoprofile of the SHEDs was analyzed using flow cytometry. Cell proliferation was assessed by the MTT reduction assay. Gene expressions of mesenchymal stem cell markers (OCT4, Nestin, CD90, and CD105) were assessed by RT-qPCR 48 h after PBMT. Data were compared by analysis of variance (ANOVA) and Tukey's test (p ≤ 0.05). Cells cultured under nutritional deficit and treated with PBMT at 5 J/cm2 presented similar cell growth than those of positive control group. Cell growth was significantly higher than those of other groups. Mesenchymal stem cell gene markers were still expressed after PBMT at 5 J/cm2. In a short-term analysis, PBMT increases the number of stem cells with no interference in the undifferentiated state of the irradiated cells, which opens wide possibilities for application in tissue regeneration.

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth.

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm2-16 s; 1.0 J/cm2-33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal-Wallis and Mann-Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm2 promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm2 groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm2) promote the proliferation of SHEDs and the maintenance of cell viability.

Axonal Degeneration in Dental Pulp Precedes Human Primary Teeth Exfoliation.

The dental pulp in human primary teeth is densely innervated by a plethora of nerve endings at the coronal pulp-dentin interface. This study analyzed how the physiological root resorption (PRR) process affects dental pulp innervation before exfoliation of primary teeth. Forty-four primary canine teeth, classified into 3 defined PRR stages (early, middle, and advanced) were fixed and demineralized. Longitudinal cryosections of each tooth were stained for immunohistochemical and quantitative analysis of dental pulp nerve fibers and associated components with confocal and electron microscopy. During PRR, axonal degeneration was prominent and progressive in a Wallerian-like scheme, comprising nerve fiber bundles and nerve endings within the coronal and root pulp. Neurofilament fragmentation increased significantly during PRR progression and was accompanied by myelin degradation and a progressive loss of myelinated axons. Myelin sheath degradation involved activation of autophagic activity by Schwann cells to remove myelin debris. These cells expressed a sequence of responses comprising dedifferentiation, proliferative activity, GAP-43 overexpression, and Büngner band formation. During the advanced PRR stage, increased immune cell recruitment within the dental pulp and major histocompatibility complex (MHC) class II upregulation by Schwann cells characterized an inflammatory condition associated with the denervation process in preexfoliative primary teeth. The ensuing loss of dental pulp axons is likely to be responsible for the progressive reduction of sensory function of the dental pulp during preexfoliative stages.

A scanning electron microscopic study of the patterns of external root resorption under different conditions.

OBJECTIVE: The aim of this study was to examine if there are qualitative differences in the appearance of external root resorption patterns of primary teeth undergoing physiologic resorption and permanent teeth undergoing pathological root resorption in different conditions. MATERIAL AND METHODS: A total of 40 teeth undergoing external root resorption in different conditions were divided into 4 groups and prepared for examination under scanning electron microscopy at magnifications ranging from 20x to 1000x. Group I: 10 primary molars exfoliated due to physiologic root resorption; Group II: 10 permanent teeth with periapical granulomas showing signs of resorption; Group III:10 permanent teeth therapeutically extracted during the course of orthodontic therapy with evidence of resorption, and Group IV: 10 permanent teeth associated with odontogenic tumors that showed evidence of resorption. RESULTS: In Group I, the primary teeth undergoing resorption showed smooth extensive and predominantly regular areas reflecting the slow ongoing physiologic process. In Group II, the teeth with periapical granulomas showed the resorption was localized to apex with a funnel shaped appearance in most cases. Teeth in Group III, which had been subjected to a short period of light orthodontic force, showed the presence of numerous resorption craters with adjoining areas of cemental repair in some cases. Teeth associated with odontogenic tumors in Group IV showed many variations in the patterns of resorption with extensive loss of root length and a sharp cut appearance of the root in most cases. CONCLUSION: Differences were observed in the patterns of external root resorption among the studied groups of primary and permanent teeth under physiologic and pathological conditions.

A scanning electron microscopic study of the patterns of external root resorption under different conditions

OBJECTIVE: The aim of this study was to examine if there are qualitative differences in the appearance of external root resorption patterns of primary teeth undergoing physiologic resorption and permanent teeth undergoing pathological root resorption in different conditions. MATERIAL AND METHODS: A total of 40 teeth undergoing external root resorption in different conditions were divided into 4 groups and prepared for examination under scanning electron microscopy at magnifications ranging from 20x to 1000x. Group I: 10 primary molars exfoliated due to physiologic root resorption; Group II: 10 permanent teeth with periapical granulomas showing signs of resorption; Group III:10 permanent teeth therapeutically extracted during the course of orthodontic therapy with evidence of resorption, and Group IV: 10 permanent teeth associated with odontogenic tumors that showed evidence of resorption. RESULTS: In Group I, the primary teeth undergoing resorption showed smooth extensive and predominantly regular areas reflecting the slow ongoing physiologic process. In Group II, the teeth with periapical granulomas showed the resorption was localized to apex with a funnel shaped appearance in most cases. Teeth in Group III, which had been subjected to a short period of light orthodontic force, showed the presence of numerous resorption craters with adjoining areas of cemental repair in some cases. Teeth associated with odontogenic tumors in Group IV showed many variations in the patterns of resorption with extensive loss of root length and a sharp cut appearance of the root in most cases. CONCLUSION: Differences were observed in the patterns of external root resorption among the studied groups of primary and permanent teeth under physiologic and pathological conditions.

Apoptosis in pulp elimination during physiological root resorption in human primary teeth.

Pulp samples of 50 healthy human teeth with indication for extraction were examined to evaluate the role of apoptosis in pulp elimination during physiological root resorption. Two groups were formed: a test group (n=30) composed of pulp samples of primary teeth with physiological root resorption and a control group (n=20) composed of pulp samples of permanent maxillary third molars. Morphological evidence of apoptosis as well as in situ detection of cellular DNA fragmentation by TUNEL assay and detection of internucleosomal pattern of fragmentation of the genomic DNA by electrophoresis were observed. The apoptotic index of the primary tooth group was significantly higher than that of the permanent tooth group (51.01 +/- 0.52 versus 25.32 +/- 0.68) (p<0.001). TUNEL reaction showed intense and diffuse labeling in the pulp samples of primary teeth, which were discrete in the controls. Intense DNA internucleosomal fragmentation, a specific pattern for apoptosis, was observed in primary tooth pulps DNA by electrophoresis, in the permanent tooth pulps this pattern fragmentation of the genomic DNA for apoptosis were not present. These results seem to indicate a role of apoptosis in pulp elimination during the physiological root resorption of human primary teeth.

Apoptosis in pulp elimination during physiological root resorption in human primary teeth

Pulp samples of 50 healthy human teeth with indication for extraction were examined to evaluate the role of apoptosis in pulp elimination during physiological root resorption. Two groups were formed: a test group (n=30) composed of pulp samples of primary teeth with physiological root resorption and a control group (n=20) composed of pulp samples of permanent maxillary third molars. Morphological evidence of apoptosis as well as in situ detection of cellular DNA fragmentation by TUNEL assay and detection of internucleosomal pattern of fragmentation of the genomic DNA by electrophoresis were observed. The apoptotic index of the primary tooth group was significantly higher than that of the permanent tooth group (51.01 ± 0.52 versus 25.32 ± 0.68) (p<0.001). TUNEL reaction showed intense and diffuse labeling in the pulp samples of primary teeth, which were discrete in the controls. Intense DNA internucleosomal fragmentation, a specific pattern for apoptosis, was observed in primary tooth pulps DNA by electrophoresis, in the permanent tooth pulps this pattern fragmentation of the genomic DNA for apoptosis were not present. These results seem to indicate a role of apoptosis in pulp elimination during the physiological root resorption of human primary teeth.

Cinqüenta amostras de polpas de dentes humanos hígidos com indicação para extração foram estudadas a fim de verificar a participação da apoptose na eliminação pulpar durante a reabsorção radicular fisiológica. As amostras foram divididas em 2 grupos: um grupo de estudo composto por 30 polpas de dentes decíduos hígidos com reabsorção radicular fisiológica, e um grupo controle composto por 20 polpas de terceiros molares superiores hígidos. Evidências morfológicas de apoptose, bem como detecção in situ da fragmentação do DNA genômico via reação de TUNEL e também a detecção do padrão internucleossômico de fragmentação do DNA genômico via eletroforese foram observados. O índice apoptótico foi maior no grupo de dentes decíduos (51,01 ± 0,52) quando comparado ao grupo de dentes permanentes (25,32 ± 0,68) (p<0,001). Quanto à reação de TUNEL, houve intensa marcação positiva para fragmentação do genoma no grupo de estudo, o que ocorreu de maneira discreta nos controle. A eletroforese do DNA genômico mostrou fragmentação internucleossômica, em um padrão específico de apoptose nas amostras de dentes decíduos o que não ocorreu no grupo de dentes permanentes. Estes achados parecem indicar a apoptose como um mecanismo importante na eliminação do tecido pulpar durante a reabsorção radicular fisiológica de dentes decíduos humanos.

A histological study of root-resected and root-transected rat incisors when eruption ceases, shortly before they are exfoliated from the socket.

Resection of the odontogenic region or root transection of normal (impeded) rat lower incisors showed that eruption ceased from 1 to 13 weeks when the base of the resected teeth (87.5%) or of the distal segment of the transected ones (86%) reached the alveolar-crest region. When the operated teeth reached the crestal region, the enamel-related periodontal tissues were absent and the periodontal ligament (PDL) was the only periodontal tissue that remained. The PDL of the crestal region may be considered as mature PDL, showing a length of approx. 5-6 6 mm at the mesial face of the tooth, 4-5 mm at lingual face and 1 mm at distal face; from these limits towards the apical end of the socket the PDL becomes gradually immature. The mature PDL seems not to have a role in the process of tooth eruption. Several factors can be suggested to explain the more frequent retention, at the crestal region of the socket, of impeded rather than unimpeded incisors submitted to the same procedures. The connective tissue that develops between the base of the tooth and the bone that fills the alveolus may have more time to organize itself in impeded than in unimpeded teeth, which erupt at a faster rate; this tissue could support and retain the impeded operated teeth longer than the unimpeded ones. The decrease in the mechanical properties of the PDL in the unimpeded condition may ease the traumatic effects and lead to exfoliation. Eruption might be stopped by the increase in occlusal forces, per unit area of root surface, as the root becomes shorter; this effect is likely to be greater in impeded than unimpeded teeth.