探討在鈦金屬表面上以不同表面處理對成骨細胞行為的影響

Investigated osteoblastic cell behavior on different treated surfaces of titanium

羅揚(Yang Lo)

指導教授 ： 陳弘森

高雄醫學大學/口腔醫學院/口腔衛生科學研究所碩士班/碩士(2012年)

摘要

由於植體表面不同的表面處理方式會影響到骨細胞的貼附和分化的表現，現今許多研究皆是在鈦植體表面上進行物理或化學改質，如：改變植體表面的親疏水性、表面電荷與粗糙度…等，目前廣泛使用的表面處理方式為噴砂加酸蝕 (sandblast and acid etching, SLA)，目前也有研究學者針對鈦植體的表面進行一連串的化學性質的改質，像是在鈦植體做表面矽烷化(Silanization)的處理，或者接枝上不同的生物聚合分子如：peptide、fibronectin、sodium styrene sulfonate…等，然而我們希望藉由複合多種的表面處理方式進一步去增強植體的錨定能力，期望可以達到更快、更好的骨整合效果。
本研究欲探討在鈦金屬表面進行不同的表面處理，對小鼠前驅骨母細胞(D1)細胞行為之影響，期望找出擁有較佳骨礦化能力的表面處理方式。
本研究中，主要使用矽烷胜肽處理(Silane-Peptide treatment)和磷酸鈣鹽的披覆處理。表面物化性分析部分，先以金相顯微鏡與掃描式電子顯微鏡(SEM) 觀察試片，並進行水滴接觸角的測試。再利用表面粗糙度測試機量測試片中心線平均粗糙度(Ra)。將試片與細胞濃度為1×105 cells/ well D l cell做接觸性的培養，培養時間為1day、4 day、7 day、10day、14day 、21day 。利用alamar blue proliferation assay 測試細胞長期增生情形；用p-Nitrophenyl phosphate (pNPP) 與染色試劑測試其ALP (Alkaline Phosphatase)之定量與定性測試。
相較於其他組別，試片在經過矽烷、胜肽處理後有最低的粗糙度(MSP: 0.09μm，SLASP: 1.12μm)和接觸角(MSP: 67˚，SLASP: 63˚)。而經過TTCP披覆的試片則有較為平坦但最疏水的表面(TTCP: 123˚)。
在細胞種植四天內，所有的組別在增生分析中沒有達顯著性的差異(p>0.05)。但在隨後的第四天和第七天，有經過SLA及矽烷胜肽處理的組別(SLASP)有著較高的表現量(p<0.0001)。ALP定量分析中，所有組別在第七天至第十天之間的ALP表現量都有相同的趨勢。ALP表現量會大量增加並持續至第二十一天，達到本實驗中ALP的最高表現量，這可能可以作為D1細胞分化的證據。此外，D1細胞在不同材料表面上分別培養一小時、一天和四天。在細胞貼附早期，細胞種植一個小時以後，各組的細胞型態多呈現圓形，但經過矽烷胜肽處理的組別可以看見些許不規則且平坦的細胞貼附型態。在細胞貼附後期，D1細胞在胜肽處理的組別有更清楚的梭形型態，特別是細胞有著漂亮的延伸形狀。而各組在第一天和第四天都可以看見由肌動蛋白細胞骨架所形成的偽足。本篇研究中發現，經過矽烷胜肽處理後的親水性表面在早期細胞貼附具有較良好的貼附型態，如果再加上3D孔洞結構的表面形貌則可得到較佳的細胞增生和分化的情形，所以若是將植體原有的SLA表面再接枝生物活性分子，則可得到較佳骨礦化能力的表面處理方法。

關鍵字

鈦 ； 表面處理 ； 胜肽

並列摘要

The major challenge for dental implants is achieving optimal esthetic appearance. A novel concept to fulfill this criterion is evaluated. Different levels of roughness can be attained by sand-blasting and acid-etching (SLA). The silane and silane with peptide treated with and without SLA treatment on the titanium surface were used as a culture substitute.
This aim of this study is investigated osteoblastic (D1) behavior on the surfaces of titanium discs with different surface treatment.
In this study, the surfaces of titanium discs treated with silanization and peptide (Silane-Peptide treatment) and coated calcium phosphate on the surface. Physicochemical analysis observed by metallographic microscope, scanning electron microscopy (SEM), and contact angle analysis. We also used the roughness machine to measure the surface roughness index. The cell viabilities were measured at time points of 1, 4, 7,10,14 and 21 days after an initial seeding of 1 × 105 D1 cells on the surface. An alamar Blue cell viability assay kit provided a simple method to count live cells by metabolic activity with using an absorbance reader. Alkaline phosphatise (ALP) production quantitative and qualitative analysis on the surface of different sample groups was determined using p-Nitrophenyl phosphate tablets and the TRACP & ALP double-stain kit.
The samples with silanization/peptide processes had lowest roughness (MSP: 0.09μm, SLASP: 1.12μm) and contact angle (MSP:67˚, SLASP:63 ˚) on their surfaces than other groups. The samples with TTCP coated processes had most hydrophobic surface (TTCP:123˚) and relatively smooth surface than other groups.

Regardless of the different surface treatments, the cell viability of the titanium disc was negligible toward D1 cells within 4 days incubation (p>0.0001).However, the surface treatment through SLA with peptide (SLASP) had the significant larger metabolic activity of D1 cells among all groups at the 4 and 7 incubation (p<0.05).
Quantitation of the ALP production on the surface of different sample groups revealed a same tendency with obvious increase on day 7 to day 10 of D1 cells culture. This expression progressively increased to day 21 and reached the maximum ALP activity could be the evidence of D1 cell differentiation.
Additionally, the fixed morpholohy of D1 cells were measured after culturing at different times of 1 h, 1 day and 4 days on the different substrates. At 1 h of cell attachment, most of fully spread cells were round morphologies, but only a few cells had an irregular and flat shape when cells adhere on treatment group. After the early stages of cell adhesion, D1 cells proliferated on the surface modification with peptide was clearly more polarized shape, especially the well cell extension was indicated in the group of peptide treated. At the proliferation stages of 1 day and 4 days cultures, the lamellipodia support by the circumferential actin cytoskeleton were formed.
This study found that good adhesion pattern in the hydrophilic surface after the Silane-peptide treatment in the early stage of cell attachment, if coupled with the 3D pore structure of the surface morphology can get a better cell proliferation and differentiation, so if the original SLA implant surface then graft bioactive molecules, may can get better surface treatment methods for promote bone mineralization.

並列關鍵字

Titanium ； Surface treatment ； Peptide

參考文獻

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