Isolation and culture of stem cells derived from dental pulp of permanent teeth: a successful method / Isolation and culture of stem cells derived from dental pulp of permanent teeth: a successful method

Objective: To establish cultures of cells from the pulp of permanent teeth by the explant method assessing parameters usually presented by stem cells, such as the expression of certain markers and the differentiation ability into osteogenic, adipogenic, and chondrogenic lineages. This study also aimed to assess the expression of ALDH1 (aldehyde dehydrogenase 1) enzyme activity on the isolated cells. Materials and method: The pulp tissue, obtained from wisdom teeth, was placed in a 6-well plate containing proper culture medium, and stored at 37 °C and 5% CO2 for cell proliferation and plastic adherence. Cells were tested for the expression of surface markers and for ALDH1 enzyme activity, by flow cytometry. In addition, cells were assessedfor multi-differentiation potential. Results: The isolated cells showed high expression of CD44 (98.8%), CD73 (100%), and CD90 (97.2%), and moderate expression of STRO-1 (18.4%) and ALDH1 (16.2%), by flow cytometry. Similarly, the cells showed differentiation ability into all three lineages of cells tested. Conclusion: Our results suggest that the explant method - or cell proliferation method - is suitable for the isolation and cultureof stem cells from dental pulp of permanent teeth. The isolated cells may be considered stem cells, based on the current criteria for their characterization, such as plastic adherence, expression of certain markers, and the absence of others, as well as multi-differentiation potential, which showed to be promising for the application in tissue regeneration.

Evaluation of laser phototherapy in the inflammatory process of the rat's TMJ induced by carrageenan.

AIM: The aim of this study was to evaluate, by light microscopy, the effects of laser phototherapy (LPT) at 780 nm or a combination of 660 and 790 nm, on the inflammatory process of the rat temporomandibular joint (TMJ) induced by carrageen. BACKGROUND: Temporomandibular disorders (TMDs) are frequent in the population and generally present an inflammatory component. Previous studies have evidenced positive effects of laser phototherapy on TMDs. However, its mechanism of action on the inflammation of the TMJ is not known yet. MATERIALS AND METHODS: Eighty-five Wistar rats were divided into 9 groups: G1, Saline; G2, Saline + LPT IR; G3, Saline + LPT IR + R; G4, Carrageenan; G5, Carrageenan + LPT IR; G6, Carrageenan + LPT IR + R; G7, previous LPT + Carrageenan; G8, previous LPT + carrageenan + LPT IR; and G9, previous LPT + carrageenan + LPT IR + R, and then subdivided in subgroups of 3 and 7 days. After animal death, specimens were taken, routinely cut and stained with HE, Sirius Red, and Toluidine Blue. Descriptive analysis of components of the TMJ was done. The synovial cell layers were counted. RESULTS: Injection of saline did not produced inflammatory reaction and the irradiated groups did not present differences compared to nonirradiated ones. After carrageenan injection, intense inflammatory infiltration and synovial cell layers proliferation were observed. The infrared irradiated group presented less inflammation and less synovial cell layers number compared to other groups. Previous laser irradiation did not improve the results. CONCLUSION: It was concluded that the LPT presented positive effects on inflammatory infiltration reduction and accelerated the inflammation process, mainly with IR laser irradiation. The number of synovial cell layers was reduced on irradiated group.

Biomodulative effects of visible and IR laser light on the healing of cutaneous wounds of nourished and undernourished Wistar rats.

AIM: We evaluated histologically the differences in the healing of cutaneous wounds on nourished or undernourished rats following laser phototherapy (LPT) (20 or 40 J/cm2). BACKGROUND: Wound healing occurs due to a competitive mechanism between the synthesis and lysis of collagen. Any factor that increases the lysis or reduces the synthesis of collagen may change the healing process. Nutritional deficiencies have a great effect on wound healing. METHODS: Fifty nourished or undernourished Wistar rats had one standardized wound created on the dorsum and were divided into 10 subgroups: Control (standard diet); Control [Northeastern Brazilian basic diet (DBR)]; Standard diet+LPT (lambda635 nm; spatial average energy fluence (SAEF) 20 J/cm2); Standard diet+LPT (lambda635 nm; SAEF 40 J/cm2); Standard diet+LPT (lambda780 nm; SAEF 20 J/cm2); Standard diet+LPT (lambda780 nm; SAEF 40 J/cm2); DBR+LPT (lambda635 nm; SAEF 20 J/cm2); DBR+LPT (lambda635 nm; SAEF 40 J/cm2); DBR+LPT (lambda780 nm; SAEF 20 J/cm2); DBR+LPT (lambda780 nm; SAEF 40 J/cm2). The first application of the treatment was carried out immediately and repeated daily for seven days. The specimens were routinely processed to wax, cut, stained with H&E and Sirius Red stains, and analyzed using light microscopy. The analyses included re-epithelization, inflammatory infiltrate, and fibroblastic proliferation. Sirius Red stained slides were used to perform descriptive analyses of the collagen fibers. RESULTS: The results showed that LPT with lambda635 wavelength was more effective for the treatment of undernourished subjects, treated with either LPT with lambda730 nm with SAEF of 20 J/cm2 or lambda780 nm with SAEF of 40 J/cm2. CONCLUSION: Nutritional status influenced the progression of the healing process as well as the quality of the healed tissue. In addition, the use of both wavelengths resulted in a positive biomodulatory effect on both nourished and undernourished subjects.

Selamento de cimentos provisórios em endodontia / Sealing of temporary cements in endodontics

Objetivo: Avaliar a microinfiltração coronária de três materiais restauradores temporários utilizados em Endodontia: Bioplic® (Biodinâmica, Londrina, Brasil), IRM® (Dentsply, Petrópolis, Brasil) e Coltosol® (Vigodent, Bonsucesso, Brasil). Métodos: Foram utilizados quarenta pré-molares inferiores íntegros, extraídos, divididos em quatro grupos: Grupo I - material restaurador provisório fotopolimerizável Bioplic® (Biodinâmica, Londrina, Brasil) associado a sistema adesivo; Grupo II - Bioplic® (Biodinâmica, Londrina, Brasil); Grupo III - material restaurador provisório à base de óxido de zinco e eugenol IRM® (Dentsply, Petrópolis, Brasil); Grupo IV - material restaurador provisório pronto para uso Coltosol® (Vigodent, Bonsucesso, Brasil). Os dentes foram imersos em solução de Rodamina B 1% e armazenados em estufa a 37ºC por 24 horas, procedendo-se à ciclagem térmica por sete dias. As temperaturas na câmara variaram em 5, 37 e 50ºC. Após a secção longitudinal dos dentes no sentido vestíbulo-lingual, a infiltração foi medida em milímetros e analisada estatisticamente através da ANOVA e teste de Tukey, com um nível de significância de 5%. Resultados: No grupo em que foi utilizada a técnica do Bioplic® (Biodinâmica, Londrina, Brasil) associado ao ataque ácido foram observados menores índices de infiltração (0,37 ± 0,24), detectando-se diferença estatisticamente significante (p<0,05) entre os grupos I e III, I e IV, II e III. Conclusão: Desta forma, todos os materiais testados apresentaram infiltração coronária, entretanto, com a utilização do Bioplic® (Biodinâmica, Londrina, Brasil) associado ao ataque ácido, foram observados os menores índices, demonstrando assim sua efetividade.

Objective: The aim of this study was to assess the coronal leakage of three temporary cements in endodontics: Bioplic® (Biodinâmica, Londrina, Brazil), IRM® (Dentsply, Petrópolis, Brazil) and Coltosol® (Vigodent, Bonsucesso, Brazil). Methods: Forty human pre-molars were divided into four groups: I (Bioplic® + adhesive system), II (Bioplic®, Biodinâmica, Londrina, Brasil), III (IRM®, Dentsply, Petrópolis, Brasil), and IV (Coltosol®, Vigodent, Bonsucesso, Brasil). The teeth were immersed in 1% Rodamine and kept at 37ºC for 24h. They were then thermal cycled for seven days. Temperatures ranged between 5, 37 and 50ºC. After longitudinal sectioning the leakage was measured in mm and statistical analysis was performed using the ANOVA and Tukey tests with a level of significance of 5%. Results: In the group in which Bioplic® was used with adhesive, a lower level of leakage was detected (0.37 ± 0.24). There was significant difference (p<0.05) between groups I and III, I and IV, II and III. Conclusion: It was concluded that all the tested material showed coronal leakage and that the use of the (Bioplic® Biodinâmica, Londrina, Brasil) plus adhesive system showed the lowest level of leakage.

Bone repair following bone grafting hydroxyapatite guided bone regeneration and infra-red laser photobiomodulation: a histological study in a rodent model.

The aim of the investigation was to assess histologically the effect of laser photobiomodulation (LPBM) on a repair of defects surgically created in the femurs of rats. Forty-five Wistar rats were divided into four groups: group I (control); group II (LPBM); group III (hydroxyapatite guided bone regeneration; HA GBR); group IV (HA GBR LPBM). The animals in the irradiated groups were subjected to the first irradiation immediately after surgery, and it was repeated every day for 2 weeks. The animals were killed 15 days, 21 days and 30 days after surgery. When the groups irradiated with implant and membrane were compared, it was observed that the repair of the defects submitted to LPBM was also processed faster, starting from the 15th day. At the 30th day, the level of repair of the defects was similar in the irradiated groups and those not irradiated. New bone formation was seen inside the cavity, probably by the osteoconduction of the implant, and, in the irradiated groups, this new bone formation was incremental. The present preliminary data seem to suggest that LPMB therapy might have a positive effect upon early wound healing of bone defects treated with a combination of HA and GBR.

The use of light photobiomodulation on the treatment of second-degree burns: a histological study of a rodent model.

OBJECTIVE: The aim of this investigation was to compare, by light microscopy, the effects of the use of laser photobiomodulation (LPBM) and polarized light (PL) on second-degree burns on rodents. BACKGROUND DATA: Burns are severe injuries that result in the loss of tissue fluids, destruction of tissues, infection, and shock. With severe and widespread third-degree burns death may occur. Several light sources have been suggested as being effective for improving wound healing. MATERIALS AND METHODS: Forty five rats were used in this study. A second-degree burn was created on the dorsum of each animal, and the animals were divided into four groups: PL (400-2000 nm, 40 mW, 2.4 J/cm(2)/min); LPBM-1 (780 nm, 35/40 mW, theta approximately 2 mm, 4 x 5 J/cm(2)); LPBM-2 (660 nm, 35/40 mW, theta approximately 2 mm, 4 x 5 J/cm(2)); and untreated animals acted as controls. The treatment was started immediately post-burn at four points around the burned area (laser: 5 J/cm(2) per site). The illumination with PL was performed according to the manufacturer's instructions. Treatments were repeated at 24-h intervals for 7 d. The animals were sacrifice at 3, 5, and 7 d post-burn. The specimens were routinely cut and stained and analyzed by light microscopy using hematoxylin and eosin and Sirius red. RESULTS: The analysis of the results demonstrated that the damaged tissue was able to efficiently absorb and process the light at all tested wavelengths. LPBM at 660 nm showed better results at early stages of wound healing. However, the use of 780-nm laser light had beneficial effects throughout the experimental period, with the animals growing newly-formed tissue similar to normal dermis. CONCLUSION: Despite our findings that the use of both types of light energy improved the healing of second-degree burns at the early stages, long-term assessment is needed to verify if this improvement will influence the final results of treatment.

Infrared laser light further improves bone healing when associated with bone morphogenetic proteins and guided bone regeneration: an in vivo study in a rodent model.

OBJECTIVE: This study assessed histologically the effect of laser photobiomodulation on the repair of surgical defects created in the femurs of Wistar rats treated or not treated with bone morphogenetic proteins (BMPs) and organic bovine bone graft. BACKGROUND DATA: This paper is part of an ongoing series of works in which biomaterials and/or guided bone regeneration (GBR) are used in association with laser photobiomodulation. Several previous reports from our group have shown that the use of laser photobiomodulation improves the treatment of bone defects. MATERIALS AND METHODS: Forty-eight adult male Wistar rats were divided into four randomized groups: group 1 (controls, n = 12); group 2 (laser photobiomodulation, n = 12); group 3 (BMPs + organic bovine bone graft + GBR, n = 12); and group 4 (BMPs + organic bovine bone graft + GBR + laser photobiomodulation, n = 12). The irradiated groups received seven irradiations every 48 h, the first immediately after the surgical procedure. Laser photobiomodulation (830 nm, 40 mW, CW, phi approximately 0.6 mm) consisted of a total of 16 J/cm2 per session at four points (4 J/cm2 each) equally spaced around the periphery of the defect. The animals were sacrificed after 15, 21, and 30 d, and the specimens were routinely embedded in wax and stained with hematoxylin and eosin and Sirius red stains and analyzed under light microscopy. RESULTS: The results showed histological evidence of increased deposition of collagen fibers (at 15 and 21 d), as well as an increased amount of well-organized bone trabeculi at the end of the experimental period (30 d) in irradiated animals compared to non-irradiated controls. CONCLUSION: We concluded that the use of laser photobiomodulation in association with BMPs, organic bovine bone grafts, and GBR increases the positive biomodulating effects of laser energy.

Infrared laser light further improves bone healing when associated with bone morphogenic proteins: an in vivo study in a rodent model.

OBJECTIVE: This study assessed histologically the effect of laser photobiomodulation (LPBM) on the repair of surgical defects created in the femurs of Wistar rats treated or not treated with bone morphogenic proteins (BMPs) and organic bovine bone graft. BACKGROUND DATA: This paper is part of an ongoing series of works in which biomaterials are used in association with LPBM. Several previous reports by our group have shown that the use of laser photobiomodulation improves the treatment of bone defects. MATERIALS AND METHODS: Forty-eight adult male Wistar rats were divided into four randomized groups: group I (control, n = 12); group II (LPBM, n = 12); group III (BMPs + organic bovine bone graft, n = 12); and group IV (BMPs + organic bovine bone graft + LPBM, n = 12). The irradiated groups received seven irradiations every 48 h, beginning immediately after the surgical procedure. The laser therapy (lambda = 830 nm, 40 mW CW, varphi = 0.6 mm) consisted of 16 J/cm(2) per session divided equally over four points (4 J/cm(2) each) around the defect. The subjects were sacrificed after 15, 21, and 30 d, and the specimens were routinely embedded in wax, stained with hematoxylin and eosin and sirius red, and analyzed under light microscopy. RESULTS: The results showed histological evidence of increased deposition of collagen fibers (at 15 and 21 d), as well as an increased amount of well-organized bone trabeculae at the end of the experimental period (30 d) in the irradiated animals versus the non-irradiated controls. CONCLUSION: The use of LPBM with BMPs and organic bovine bone grafts increases the positive biomodulating effects of laser light.

Avaliação do emprego da fototerapia laser no processo inflamatório induzido por carragenina na articulação temporomandibular de rato / Evaluation of laser phototherapy in the inflammatory process of the rats temporomandibular joint induced by carrageenon

As disfunções temporomandibulares (DTMs) são freqüentes na população egeralmente estão associadas a processos inflamatórios. Estudos anteriores têm evidenciado efeitos positivos da fotobiomodulação laser (FBML) nas DTMs. Contudo, seu mecanismo de ação na resposta inflamatória da articulação temporomandibular (ATM) ainda é pouco conhecido. O objetivo deste trabalho foi avaliar através da análise histológica a ação da Fototerapia Laser - FTL (GaAlAs, λ780nm - IV, P=50mW, CW, φ~4mm2, 10J/cm2 por sessãoisoladamente, ou associado ao λ660nm - V, P=40mW, 5J/cm2 cada λ) naresposta inflamatória da ATM de ratos induzida por carragenina. Oitenta ecinco ratos Wistar foram divididos em nove grupos: G1 – Solução salina; G2 –Solução salina + FTL IV; G3 – Solução salina + FTL IV+V; G4 – Carragenina;G5 – Carragenina + FTL IV; G6 – Carragenina + FTL IV+V; G7 – Laser prévio + carragenina; G8 – Laser prévio + carragenina + FTL IV e G9 – Laser prévio + carragenina + FTL IV+V, que foram subdivididos em subgrupos de três e sete dias. Após o sacrifício, os espécimes de tecidos foram processados e corados com HE, Picrosirius e Azul de Toluidina. Foi realizada análise descritiva das estruturas que compõem a ATM, observação da expressão de colágeno no côndilo e disco articular e contagem das camadas de células da membrana sinovial. Nos grupos em que foi injetada solução salina não foi observada reação inflamatória e os grupos irradiados não apresentaram diferença em relação ao grupo não irradiado. Após injeção de carragenina, foram observados intenso infiltrado inflamatório e proliferação do número de camadas da membrana sinovial. O grupo irradiado com laser IV apresentou menor inflamação e menor número de camadas nos dois períodos de observação comparado a todos os grupos. A aplicação do laser prévio não promoveu melhora nos resultados finais. Conclui-se que a FTL apresentou efeitos positivos em relação à redução do infiltrado inflamatório e aceleração doprocesso de...

Biomodulative effects of polarized light on the healing of cutaneous wounds on nourished and undernourished Wistar rats.

OBJECTIVE: This study aimed to evaluate, by light microcopy, the differences in healing process of cutaneous wounds on nourished or undernourished rats following illumination by polarized light (lambda400-2000 nm) with 20 or 40 J/cm(2). BACKGROUND DATA: There are some reports in the literature on different effects of polarized light on wound healing. Amongst the factors that interfere with wound healing one is the nutritional status of the subject. METHODS: Thirty nourished or undernourished Wistar rats had one standardized surgical wound created on the dorsum and were divided into six groups: group 1, control (standard diet); group 2, control (Northeastern Brazilian Basic Diet [DBR]); group 3, standard diet + polarized light (20 J/cm(2)); group 4, standard diet + polarized light (40 J/cm(2)); group 5, DBR + polarized light (20 J/cm(2)); group 6, DBR + polarized light (40 J/cm(2)). The first application of treatment was carried out immediately after wounding and repeated every 24 h during 7 days. The animals were sacrificed, and specimens were taken and routinely processed to wax, cut, and stain with hematoxylin and eosin (H&E) and Sirius Red. These were then analyzed under light microscopy. The analysis included re-epitheliialization, inflammatory infiltrate, and fibroblastic proliferation. Sirius Red-stained slides were used to perform descriptive analysis of collagen. RESULTS: The analysis of the results showed better results in these groups illuminated with 20 J/cm(2). CONCLUSION: It is concluded that nutritional status influenced the progression of the healing process as well as the quality of the healed tissue, and that the use of polarized light resulted in a positive biomodulatory effect.

Phototherapy improves healing of cutaneous wounds in nourished and undernourished Wistar rats.

A wound represents the interruption of the continuity of tissue that is followed by damage or cellular death. Wound healing occurs due to a competitive mechanism between the synthesis and lysis of collagen. Any factor that increases collagen lysis or reduces its synthesis may result in changes in the healing process, i.e., nutritional deficiencies. Phototherapies have been suggested as an effective method to improve wound healing. This study evaluated, histologically, the differences in the healing of cutaneous wounds in nourished and undernourished rats following laser therapy or illumination by polarized light. Fifty nourished or undernourished Wistar rats had a standardized wound created on the dorsum and were divided into 6 subgroups: Group 1--Control (Standard diet; n=5); Group 2--Control (DBR; n=5); Group 3--Standard diet + laser therapy (lambda635nm; 20J/cm2, n=5; or 40J/cm2, n=5); Group 4--Standard diet + Bioptron (lambda400-2000nm; 20J/cm2, n=5; or 40 J/cm2, n=5); Group 5--DBR + laser therapy (lambda635nm; 20J/cm2, n=5; or 40J/cm2, n=5); Group 6--DBR + Bioptron (lambda400-2000nm; 20J/cm2, n=5; or 40 J/cm2, n=5). The first application of the treatment was carried out immediately after surgery and repeated every 24 h during 7 days. Specimens were routinely processed (wax, cut and stained with H&E and Picrosirius stain) and analyzed under light microscopy. Analysis included re-epithelization, inflammatory infiltrate, and fibroblastic proliferation. Picrosirius stained slides were used to perform descriptive analysis of the collagen fibers. The results showed the best results for nourished and undernourished groups treated with polarized light at a dose of 20J/cm2 and the undernourished groups irradiated with the laser light. It is concluded that the nutritional status influenced the progression of the healing process as well as the quality of the healed tissue and that the use of both modalities of phototherapy resulted in a positive biomodulatory effect in both nourished and undernourished subjects. The effect of the polarized light was more evident in nourished subjects and laser therapy more effective in the treatment of undernourished subjects, in both cases with a dose of 20J/cm2.