以不同清潔方式處理牙周病菌感染後之鈦金屬板其表面性質變化和細胞貼附情形

Abstract

本研究在第四級純鈦金屬板 (直徑15 m，厚度2 mm，粗糙度Ra=1.3 μm)上接種牙周病菌A .actinomycetemcomitans及P. gingivalis培養生物薄膜，並模擬臨床移除生物薄膜的方式分為五組，第一組不接種細菌也不進行清創，第二組接種細菌但不進行清創，第三組以鈦金屬刮匙進行清創配合PBS沖洗，第四組以鈦金屬刮匙進行清創配合PBS沖洗後進行超音波震盪，第五組以鈦金屬刮匙進行清創配合PBS沖洗後浸泡Chlorhexidine。之後以掃描式電子顯微鏡觀察A .actinomycetemcomitans及P. gingivalis是否能在鈦板形成生物薄膜，並觀察清創處理之後的細菌殘留情形，我們發現經由超音波震盪的方式可將細菌完全移除。在細胞貼附測試結果部分發現即使經由超音波震盪將細菌完全移除後細胞的貼附數目仍不如未接種細菌之鈦板，推測是表面性質有所改變或是有殘留細菌之分泌物如:內毒素等阻礙細胞之貼附。接下來進行表面分析，在粗糙度部分我們發現P. gingivalis所產生的生物薄膜會使粗糙度降低，而A. actinomycetemcomitans則不會，而經由鈦金屬刮匙進行清創會使粗糙度有下降的情形，超音波震盪與浸泡Chlorhexidine則不會。在親疏水部份我們發現即使經由超音波震盪亦無法將鈦板之親水性恢復到未感染的狀態。由於文獻指出P. gingivalis及A.actinomycetemomitans之內毒素會使骨母細胞之分化能力及增生能力下降，也對細胞之貼附性有影響。我們在鈦板上coating內毒素發現接觸角增加，因此我們推測即使以超音波震盪完全移除細菌之後其上仍殘留內毒素，是導致骨母細胞在先前的實驗中無法順利貼附的原因。因此對表面進行化學分析，而使用傅立葉轉換紅外線光譜儀及X射線光電子能譜儀都無法測得。但經由LAL試驗在超音波震盪後的鈦板測量到內毒素的殘留。因此我們認為在臨床植體周圍炎發生後之re-osseointegration效率不佳或是無法進行的原因是因植體表面殘有內毒素。

Periodontal bacteria A. actinomycetemcomitans and P. gingivalis were inoculated on commercialy grade 4 pure titanium discs (15 mm in diameter, 2 mm in thickness, and Ra=1.3) respectively to form in vitro biofilm. They were divided into five groups, Group 1 received no treatment and served as control. Group 2 received bacteria inoculation without treatment. Group 3 received bacterial inoculation, curette debridement and PBS irrigation. Group 4 received bacterial inoculation, curette debridement, PBS irrigation and ultrasonication. Group 5 received bacterial inoculation, curette debridement, PBS irrigation and chlorhexidine treatment. SEM result indicated that ultrasonication was noted to eliminate all bacteria on the titanium disc and was thought to be the most effective way to remove bacteria in this study. The result of HEPM cells adhesion assay indicated that attached cells were decreased even bacteria were completely removed by ultrasonication when compared with the titanium disc without bacteria inoculation. We thus coating endotoxin on titanium disc and showed increased in contact angle. However, endotoxin couldn’t be detected using FTIR and XPS. Limulus ameboycte lysate result showed presence of endotoxin after ultrasonication. We concluded that re-osseointegration failure after peri-implantitis is due to endotoxin residues on the implant.