LED antimicrobial photodynamic therapy with phenothiazinium dye against Staphylococcus aureus: An in vitro study.

The objective of this study was to evaluate, in vitro, the bactericidal effect of AmPDT on Staphylococcus aureus (ATCC 25923) using different concentrations (100, 50, 25, 12.5 and 6.25µg/mL) of phenothiazine compound combined with LED light (λ632±2nm) using varied energy densities (12, 9.6, 7.2, 4.8 and 2.4J/cm2). The experiments were carried out in triplicate and the samples were divided into groups: Control, Irradiated (treated only with light at different energy densities), Photosensitizer (treated only in the presence of the dye), AmPDT (treatment with light associated with dye). Counts of the colony forming units and the data obtained were statistically analyzed (ANOVA, Tukey's test, p<0.05). The results showed no difference between irradiated and Control groups. However, using the photosensitizer alone caused significant increased cytotoxicity and consequent reduction on the CFU counts (12.5µg/mL (p<0.001), 25µg/mL, 50µg/mL and 100µg/mL (p<0.0001). When AmPDT was used significant inhibition above 70% were detected for all concentrations of the photosensitize (p<0.0001) except for 6.25µg/mL. The results indicate a dose-response dependent when the photosensitizer is used alone but not for the sole use of the light is used. It is concluded that, a single application of AmPDT, using energy density of 12J/cm2 associated either to 12.5 (81.52%) or 25µg/mL (91.57%) resulted in higher in vitro inhibition of S. aureus.

Repair of surgical bone defects grafted with hydroxylapatite + ß-TCP and irradiated with λ=850 nm LED light.

The treatment of bone loss due to different etiologic factors is difficult and many techniques aim to improve the repair, including a wide range of biomaterials and recently, photobioengineering. This work aimed to assess by histological analysis the repair of bone defects grafted with biphasic synthetic micro-granular HA + ß-TCP associated with LED phototherapy. Forty rats were divided into 4 groups (Clot, LED, Biomaterial and LED + Biomaterial) each subdivided into 2 subgroups according to the time of animal death (15 and 30 days). Surgical bone defects were prepared on the femur of each animal with a trephine drill. In animals of the Clot group the defect was filled only by blood clot, in the LED group the defect filled with the clot was further irradiated. In the animals of Biomaterial and LED + Biomaterial groups the defect was filled by biomaterial and the last one was further irradiated (λ = 850 ± 10 nm, 150 mW, Φ ~ 0.5 cm2, 20 J/cm2 - session, 140 J/cm2- treatment) at 48-h intervals for 2 weeks. Following animal death, samples were taken and analyzed by light microscopy. Using the degree of maturation of the bone by assessment of the deposition/organization of the basophilic lines in the newly formed bone tissue, the LED + Biomaterial group was the one in a more advanced stage of bone repair process at the end of the experiment. It may be concluded that the use of LED phototherapy was effective in positively modulating the process of bone repair of bone defects in the femur of rats submitted or not to biomaterial grafting.

Repair of Surgical Bone Defects Grafted with Hydroxylapatite + ß-TCP and Irradiated with λ=850 nm LED Light

The treatment of bone loss due to different etiologic factors is difficult and many techniques aim to improve the repair, including a wide range of biomaterials and recently, photobioengineering. This work aimed to assess by histological analysis the repair of bone defects grafted with biphasic synthetic micro-granular HA + β-TCP associated with LED phototherapy. Forty rats were divided into 4 groups (Clot, LED, Biomaterial and LED + Biomaterial) each subdivided into 2 subgroups according to the time of animal death (15 and 30 days). Surgical bone defects were prepared on the femur of each animal with a trephine drill. In animals of the Clot group the defect was filled only by blood clot, in the LED group the defect filled with the clot was further irradiated. In the animals of Biomaterial and LED + Biomaterial groups the defect was filled by biomaterial and the last one was further irradiated (λ=850±10 nm, 150 mW, Φ ~ 0.5 cm2, 20 J/cm2 - session, 140 J/cm2- treatment) at 48-h intervals for 2 weeks. Following animal death, samples were taken and analyzed by light microscopy. Using the degree of maturation of the bone by assessment of the deposition/organization of the basophilic lines in the newly formed bone tissue, the LED + Biomaterial group was the one in a more advanced stage of bone repair process at the end of the experiment. It may be concluded that the use of LED phototherapy was effective in positively modulating the process of bone repair of bone defects in the femur of rats submitted or not to biomaterial grafting.

O tratamento de perdas ósseas devido a diferentes fatores etiológicos é difícil e muitas técnicas têm por objetivo melhorar o reparo incluindo o uso de uma ampla gama de biomateriais e, recentemente, a fotobioengenharia. Este trabalho teve como objetivo avaliar, por meio de análise histológica, o reparo de defeitos ósseos enxertados com HA bifásica micro-granular sintética + β -TCP associada à fototerapia LED. Quarenta ratos foram divididos em quatro grupos (Clot, LED, Biomaterial e LED + Biomaterial), subdivididos no dois subgrupos de acordo com o momento da morte (15 e 30 dias). Defeitos ósseos cirúrgicos foram criados em um fêmur de cada animal com uma broca trefina. Em animais do grupo coágulo, o defeito foi preenchido apenas pelo coágulo sanguíneo, no grupo de LED o defeito foi preenchido pelo coágulo e irradiado. Nos animais dos grupos do biomaterial e LED + biomaterial, os defeitos foram preenchidos com biomaterial e o último foi adicionalmente irradiado (λ=850±10 nm, 150 mW, Φ ~ 0.5 cm2, 20 J/cm2 - session, 140 J/cm2 -tratamento) a cada 48 h por duas semanas. Após a morte dos animais, amostras foram colhidas e analisadas por microscopia de luz, usando o grau de maturação do osso como marcador (deposição/organização das linhas basofílicas) no tecido ósseo neoformado. O grupo de LED + biomaterial apresentou processo de reparação mais avançado ao fim do experimento. Pode-se concluir que o uso da fototerapia LED foi eficaz na modulação positiva do processo de reparo ósseo de defeitos ósseos no fêmur de ratos submetidos ou não a enxerto com biomaterial.

Raman spectroscopic study of the repair of surgical bone defects grafted or not with biphasic synthetic micro-granular HA + ß-calcium triphosphate irradiated or not with λ850 nm LED light.

The handling of bone losses due to different etiologic factors is difficult and many techniques are aim to improve repair, including a wide range of biomaterials and, recently, photobioengineering. This work aimed to assess, through Raman spectroscopy, the level of bone mineralization using the intensities of the Raman peaks of both inorganic (~960, ~1,070, and 1,077 cm(-1)) and organic (~1,454 and ~1,666 cm(-1)) contents of bone tissue. Forty rats were divided into four groups each subdivided into two subgroups according to the time of sacrifice (15 and 30 days). Surgical bone defects were made on the femur of each animal with a trephine drill. On animals of group clot, the defect was filled only by blood clot, on group LED, the defect filled with the clot was further irradiated. On animals of groups biomaterial and LED + biomaterial, the defect was filled by biomaterial and the last one was further irradiated (λ850 ± 10 nm, 150 mW, Φ ~ 0.5 cm(2), 20 J/cm(2)-session, 140 J/cm(2)-treatment) at 48-h intervals and repeated for 2 weeks. At both 15th and 30th days following sacrifice, samples were taken and analyzed by Raman spectroscopy. At the end of the experimental time, the intensity of hydroxyapatite (HA) (~960 cm(-1)) were higher on group LED + biomaterial and the peaks of both organic content (~1,454 and ~1,666 cm(-1)) and transitional HA (~1,070 and ~1,077 cm(-1)) were lower on the same group. It is concluded that the use of LED phototherapy associated to biomaterial was effective in improving bone healing on bone defects as a result of the increasing deposition of HA measured by Raman spectroscopy.

Raman ratios on the repair of grafted surgical bone defects irradiated or not with laser (λ780 nm) or LED (λ850 nm).

This work aimed to assess biochemical changes associated to mineralization and remodeling of bone defects filled with Hydroxyapatite+Beta-Beta-tricalcium phosphate irradiated or not with 2 light sources. Ratios of intensities, band position and bandwidth of selected Raman peaks of collagen and apatites were used. Sixty male Wistar rats were divided into 6 groups subdivided into 2 subgroups (15th and 30th days). A standard surgical defect was created on one femur of each animal. In 3 groups the defects were filled with blood clot (Clot, Clot+Laser and Clot+LED groups) and in the remaining 3 groups the defects were filled with biomaterial (Biomaterial, Biomaterial+Laser and Biomaterial+LED groups). When indicated, the defects were irradiated with either Laser (λ780 nm, 70 mW, Φ∼0.4 cm(2)) or LED (λ850±10 nm, 150 mW, Φ∼0.5 cm(2)), 20 J/cm(2) each session, at 48 h intervals/2 weeks (140 J/cm(2) treatment). Following sacrifice, bone fragments were analyzed by Raman spectroscopy. Statistical analysis (ANOVA General Linear Model, p<0.05) showed that both grafting and time were the variables that presented significance for the ratios of ∼1660/∼1670 cm(-1) (collagen maturation), ∼1077/∼854 cm(-1) (mineralization), ∼1077/∼1070 cm(-1) (carbonate substitution) and the position of the ∼960 cm(-1) (bone maturation). At 30th day, the ratios indicated an increased deposition of immature collagen for both Clot and Biomaterial groups. Biomaterial group showed increased collagen maturation. Only collagen deposition was significantly dependent upon irradiation independently of the light source, being the amount of collagen I increased in the Clot group at the end of the experimental time. On the other hand, collagen I deposition was reduced in biomaterial irradiated groups. Raman ratios of selected protein matrix and phosphate and carbonate HA indicated that the use of biphasic synthetic micro-granular HA+Beta-TCP graft improved the repair of bone defects, associated or not with Laser or LED light, because of the increasing deposition of HA.

Influence of the λ780nm laser light on the repair of surgical bone defects grafted or not with biphasic synthetic micro-granular hydroxylapatite+Beta-Calcium triphosphate.

The treatment of bone loss due to different etiologic factors is difficult and many techniques aim to improve repair, including a wide range of biomaterials and, recently, photobioengineering. This work aimed to assess, through histological analysis The aim of this study was to assess, by light microscopy, the repair of bone defects grafted or not with biphasic synthetic micro-granular Calcium hydroxyapatite (HA)+Beta-TCP associated or not with Laser phototherapy - LPT (λ780nm). Forty rats were divided into 4 groups each subdivided into 2 subgroups according to the time of sacrifice (15 and 30days). Surgical bone defects were made on femur of each animal with a trephine drill. On animals of Clot group the defect was filled only by blood clot, on Laser group the defect filled with the clot was further irradiated. On animals of Biomaterial and Laser+Biomaterial groups the defect was filled by biomaterial and the last one was further irradiated (λ780nm, 70mW, spot size∼0.4cm(2), 20J/cm(2)-session, 140J/cm(2)-treatment) in four points around the defect at 48-h intervals and repeated for 2weeks. At both 15th and 30th days following sacrifice, samples were taken and analyzed by light microscopy. Many similarities were observed histologically between groups on regards bone reabsorption and neoformation, inflammatory infiltrate and collagen deposition. The criterion degree of maturation, marked by the presence of basophilic lines, indicated that the use of LPT associated with HA+Beta TCP graft, resulted in more advanced stage of bone repair at the end of the experiment.

Raman study of the repair of surgical bone defects grafted with biphasic synthetic microgranular HA + ß-calcium triphosphate and irradiated or not with λ780 nm laser.

The treatment of bone loss due to different etiologic factors is difficult, and many techniques aim to improve repair, including a wide range of biomaterials and, recently, photobioengineering. This work aimed to assess, through Raman spectroscopy, the level of bone mineralization using the intensities of the Raman peaks of both inorganic (∼ 960, ∼ 1,070, and ∼ 1,077 cm(-1)) and organic (∼ 1,454 and ∼ 1,666 cm(-1)) contents of bone tissue. Forty rats were divided into four groups each subdivided into two subgroups according to the time of killing (15 and 30 days). Surgical bone defects were made on femur of each animal with a trephine drill. On animals of group Clot, the defect was filled only by blood clot; on group Laser, the defect filled with the clot was further irradiated. On animals of groups Biomaterial and Laser + Biomaterial, the defect was filled by biomaterial and the last one was further irradiated (λ780 nm, 70 mW, Φ âˆ¼ 0.4 cm(2), 20 J/cm(2) session, 140 J/cm(2) treatment) in four points around the defect at 48-h intervals and repeated for 2 weeks. At both 15th and 30th day following killing, samples were taken and analyzed by Raman spectroscopy. At the end of the experimental time, the intensities of both inorganic and organic contents were higher on group Laser + Biomaterial. It is concluded that the use of laser phototherapy associated to biomaterial was effective in improving bone healing on bone defects as a result of the increasing deposition of calcium hydroxyapatite measured by Raman spectroscopy.

New bone formation around implants inserted on autologous and xenografts irradiated or not with IR laser light: a histomorphometric study in rabbits.

Use of biomaterials and light on bone grafts has been widely reported. This work assessed the influence of low-level laser therapy (LLLT) on bone volume (BV) and bone implant contact (BIC) interface around implants inserted in blocks of bovine or autologous bone grafts (autografts), irradiated or not, in rabbit femurs. Twenty-four adult rabbits were divided in 8 groups: AG: autograft; XG: xenograft; AG/L: autograft + laser; XG/L: xenograft + laser; AG/I: autograft + titanium (Ti) implant; XG/I: xenograft + Ti implant; AG/I/L: autograft + Ti implant + laser; and XG/I/L: xenograft + Ti implant + laser. The animals received the Ti implant after incorporation of the grafts. The laser parameters in the groups AG/L and XG/L were λ=780 nm, 70 mW, CW, 21.5 J/cm 2 , while in the groups AG/I/L and XG/I/L the following parameters were used: λ=780 nm, 70 mW, 0.5 cm 2 (spot), 4 J/cm 2 per point (4), 16 J/cm 2 per session, 48 h interval × 12 sessions, CW, contact mode. LLLT was repeated every other day during 2 weeks. To avoid systemic effect, only one limb of each rabbit was double grafted. All animals were sacrificed 9 weeks after implantation. Specimens were routinely stained and histomorphometry carried out. Comparison of non-irradiated and irradiated grafts (AG/L versus AG and XG/L versus XG) showed that irradiation increased significantly BV on both grafts (p=0.05, p=0.001). Comparison between irradiated and non-irradiated grafts (AG/I/L versus AG/I and XG/I/L versus XG/I) showed a significant (p=0.02) increase of the BIC in autografts. The same was seen when xenografts were used, without significant difference. The results of this investigation suggest that the use of LLLT is effective for enhancing new bone formation with consequent increase of bone-implant interface in both autologous grafts and xenografts.

New Bone Formation around Implants Inserted on Autologous and Xenografts Irradiated or not with IR Laser Light: A Histomorphometric Study in Rabbits

Use of biomaterials and light on bone grafts has been widely reported. This work assessed the influence of low-level laser therapy (LLLT) on bone volume (BV) and bone implant contact (BIC) interface around implants inserted in blocks of bovine or autologous bone grafts (autografts), irradiated or not, in rabbit femurs. Twenty-four adult rabbits were divided in 8 groups: AG: autograft; XG: xenograft; AG/L: autograft + laser; XG/L: xenograft + laser; AG/I: autograft + titanium (Ti) implant; XG/I: xenograft + Ti implant; AG/I/L: autograft + Ti implant + laser; and XG/I/L: xenograft + Ti implant + laser. The animals received the Ti implant after incorporation of the grafts. The laser parameters in the groups AG/L and XG/L were λ=780 nm, 70 mW, CW, 21.5 J/cm 2 , while in the groups AG/I/L and XG/I/L the following parameters were used: λ=780 nm, 70 mW, 0.5 cm 2 (spot), 4 J/cm 2 per point (4), 16 J/cm 2 per session, 48 h interval × 12 sessions, CW, contact mode. LLLT was repeated every other day during 2 weeks. To avoid systemic effect, only one limb of each rabbit was double grafted. All animals were sacrificed 9 weeks after implantation. Specimens were routinely stained and histomorphometry carried out. Comparison of non-irradiated and irradiated grafts (AG/L versus AG and XG/L versus XG) showed that irradiation increased significantly BV on both grafts (p=0.05, p=0.001). Comparison between irradiated and non-irradiated grafts (AG/I/L versus AG/I and XG/I/L versus XG/I) showed a significant (p=0.02) increase of the BIC in autografts. The same was seen when xenografts were used, without significant difference. The results of this investigation suggest that the use of LLLT is effective for enhancing new bone formation with consequent increase of bone-implant interface in both autologous grafts and xenografts.

O uso de biomateriais e luz em enxertos ósseos têm sido relatados. Esse trabalho avaliou a influência do laser baixa potência - LBP no volume ósseo (VO) e superfície de contato osso-implante (COI) ao redor de implantes dentários inseridos em blocos de enxerto bovino ou autólogos incorporados, irradiados ou não, em fêmures de coelho. Vinte e quatro coelhos adultos foram divididos em 8 grupos: EA: enxerto autólogo; EX: enxerto xenógeno; EA/L: enxerto autólogo + laser; EX/L: enxerto xenógeno + laser; EA/I: enxerto autólogo + implante; EX/I: enxerto xenógeno + implante; EA/I/L: enxerto autólogo + implante de titânio + laser; EX/I/L: enxerto xenógeno + implante de titânio + laser. Os animais receberam um implante de titânio após a incorporação dos enxertos. Os parâmetros de laser nos grupos EA/L e EX/L foram λ =780 nm, 70 mW, CW, 21,5 J/cm 2 ), enquanto que nos grupos EA/I/L e EX/I/L os seguintes parâmetros de laser foram utilizados: λ =780 nm, 70 mW, 0,5 cm 2 (spot), 4 J/cm 2 por ponto (4), 16 J/cm 2 por sessão, intervalo de 48 h × 12 sessões, CW, modo contato. O LBP foi repetido a cada 48 h (2 semanas). Para evitar efeito sistêmico apenas um membro de cada coelho foi duplamente enxertado. Todos os animais foram sacrificados 9 semanas após o implante. Os espécimes foram corados rotineiramente e histomorfometria foi realizada. A comparação dos enxertos não-irradiados e irradiados (EA/L versus EA e EX/L versus EX) mostrou que a irradiação aumentou significantemente (p=0,02) o VO para ambos os tipos de enxertos (p=0,05, p=0,001). A comparação dos enxertos não-irradiados e irradiados (EA/I/L versus EA/I e EX/I/L versus EX/I) mostrou um aumento significante (p=0,02) do COI nos enxertos autólogos e xenógenos sem diferença estatística. Os resultados desta investigação sugerem que o uso de LBP é efetivo para aumentar a neoformação óssea com consequente aumento do COI em enxertos autólogos e xenógenos.