| Abstract: | 使用抗微生物光動力療法（aPDT）的細菌消除被認為是植體周圍炎治療中的替代治療方式。在研究的第一部分中討論了與亞甲藍（MB）介導的aPDT的殺菌效果有關的幾個因素：不同的MB濃度，MB的pH和照射時間。然而，對污染的鈦合金進行殺菌清潔只是治療的首要部分。另外，應該在去污染的Ti合金表面上觀察到骨再整合效應。本研究的第一部分評估了MB介導的aPDT在噴砂，大顆粒和酸蝕刻（SLA）預處理的鈦合金消除革蘭氏陰性（P. gingivalis和A. actinomycetemcomitans）和革蘭氏陽性（S. mutans）細菌的劑量依賴性和pH依賴性殺菌作用。本研究的第二部分是在濃度依賴性使用MB-aPDT後評估污染的SLA（噴砂，大砂礫和酸蝕刻）Ti合金表面上的成骨細胞功能。
在第一部分中，不同的MB濃度（50,100和200μg/ mL），MB pH（4,7和10）和照射時間（0,30和60 s）對細菌活力的影響檢測殘留脂多醣（LPS）的含量。還檢測了在未受污染和污染的樣品上aPDT 60秒後MB溶液的pH變化。實驗結果表明，與單獨使用MB相比，MB介導的PDT可有效殺死生物膜污染鈦合金表面上的大部分細菌。值得注意的是，隨著MB濃度和照射時間的增加，aPDT表現出更好的抗菌功效。在生物膜污染的樣品上的酸性溶液中處理時，aPDT導致pH增加。相反，在aPDT後，最初的高鹼性pH降低至約pH8.5的值。有趣的是，中性pH值有微小的變化，與MB濃度和細菌種類無關。正如所料，在較高pH環境下具有較高MB濃度的aPDT顯著降低了A. actinomycetemcomitans 和 P. gingivalis的LPS濃度。
關於成骨細胞功能的第二部分中，使用雷射進行60秒照射，對粘附在樣品上的革蘭氏陰性菌或革蘭氏陽性菌進行具有不同MB濃度（200,250,300,350和400μg/ mL）的aPDT。除了殺菌功效的評估之外，另對於MB-aPDT殺菌後鈦合金表面以支持成骨細胞樣MG63附著，增殖，分化和礦化，並評估預定的培養時間間隔。重要的結果是使用具有較高MB濃度的PDT在消毒表面上培養的成骨細胞的表現與沒有污染的對照相當。在該體外模型的限制範圍內，aPDT中使用的400μg/ mL MB的這種配方不僅可以是針對受細菌污染的植入物的致死濃度，而且還可以增強受污染植入物上的成骨細胞功能。然而，在植體周圍炎治療的臨床實踐中的功效仍有待研究。
Bacterial elimination using antimicrobial photodynamic therapy (aPDT) has been considered an alternative therapeutic modality in peri-implantitis treatment. There were several factors related to the bactericidal effects of methylene blue (MB)-mediated aPDT discussed in the first part of study: different MB concentrations, the pH of the MB, and irradiation time. However, the bactericidal cleaning on the contaminated titanium is only the first part of the treatment. In addition,the re-osseointegration effect should be observed on the decontaminated Ti surface. The first part of the present study evaluated the dose-dependent and pH-dependent bactericidal effects of MB-mediated aPDT at eliminating Gram-negative (P. gingivalis and A. actinomycetemcomitans) and Gram-positive (S. mutans) bacteria on sandblasting, large-grit and acid-etching (SLA)-pretreated titanium alloy. And the second part of the present was to evaluate osteoblast functions on the contaminated SLA (sandblasting, large-grit and acid-etching) Ti alloy surfaces after the concentration-dependent use of MB-aPDT.
In the first part, the effects of different MB concentrations (50, 100, and 200 μg/mL), MB pH (4, 7, and 10), and irradiation time (0, 30, and 60 s) on the bacterial viability and residual lipopolysaccharide (LPS) levels were examined. The variations in the pH of the MB solution after aPDT for 60 s on the uncontaminated and contaminated specimens were also detected. The experimental results indicated that MB-mediated PDT could effectively kill the majority of bacteria on the titanium alloy surfaces of biofilm-contaminated implants compared with the MB alone. Of note, aPDT exhibited better antibacterial efficacy with increase in the MB concentration and irradiation time. While treated in an acidic solution on the biofilm-contaminated specimens, aPDT caused the pH to increase. By contrast, the initially high alkaline pH decreased to a value of about pH 8.5 after aPDT. Intriguingly, the neutral pH had minor changes, independent of the MB concentration and bacterial species. As expected, aPDT with higher MB concentration at higher pH environment significantly lowered the LPS concentration of A. actinomycetemcomitans and P. gingivalis.
In the second part regarding osteoblast functions, Gram-negative or Gram-positive adhered on disc samples was subjected to aPDT with different MB concentrations (200, 250, 300, 350, and 400 μg/mL) using laser for 60 s irradiation. In addition to the evaluation of elimination efficacy, the disinfected disc surfaces by MB-aPDT to support osteoblast-like MG63 attachment, proliferation, differentiation, and mineralization were assessed for the predetermined culture time intervals. The important results were that the expression of osteoblast cultured on disinfected surfaces using aPDT with higher MB concentration was comparable to the control without contamination. Within the limits of this in vitro model, this formulation of 400 μg/mL MB used in aPDT may be not only the lethal concentration against the bacteria-contaminated implants, but it could also enhance the osteoblast functions on the contaminated implants. Nevertheless, the efficacy in the clinical practice for peri-implantitis therapy remains to be studied. |
