A Preliminary Study of Osseointegration in the Dental Implant Therapy in vitro-culture of-Culture of Mouse KUSA/A1 Cells on Titanium Plates with Different Surface Modifications

人為培養環境內(in vitro)牙科植體骨整合的初步研討－鼠KUSA/A1細胞在不同表面處理的鈦板上的培養生長

中塚美智子(Michiko Nakatsuka) ； 三上淑子(Toshiko Mikami) ； 橋本典也(Yoshiya Hasimoto) ； 紺井拡隆(Hirotaka Koni) ； 陳弘森(Hong-Sen Chen) ； 蕭思郁(Szu-Yu Hsiao) ； 隈部俊二(Shunji Kumabe) ； 黃純德(Shun-Te Huang) ； 岩井康智(Yasutomo Iwai) ； 黃宏智(Hung-Chih Huang)

《Taiwan Journal of Oral Medicine Sciences》 25卷3期 (2009/12) Pp. 4-19

https://doi.org/10.7059/TJOMS.200912.0004

摘要

為了獲得成功的牙科植體治療，確實良好而廣泛的骨整合及植體－骨骼間接觸為必要條件。在現今人工培養環境中的研究，我們初步証明了KUSA/A1細胞從培養的第三天；即大量繁殖並分化為分泌骨鈣素的類成骨細胞。我們在以下三種钛及钛合金圓盤上培養類成骨細胞：(1)A.O.即陽極氧化處理。(2)Ha:羟磷灰石覆蓋。(3)S.P.I:精密噴砂處理。這三種植體的表面處理與現今普及的商用植體是一樣的。我們在培養了180分鐘後，研討了這三種植體上衍生的類成骨細胞的組織學像，用以探究微環境中模擬牙科植體骨整合的成效。在培養60分鐘時，我們採樣這三種檢體，在掃描電子顯微鏡下，可發現鬆散的球狀到卵圓狀類成骨細胞散佈著（平均直徑10μm），且向植體表面凹陷處移動。這些增生中的類成骨細胞有著腫脹的胞體，並伸出許多微小的絲狀偽足和一些片狀胞足與植體表面接觸，一些具有細胞問鍵結的類成骨細胞也零星散佈著。相對的，在培養180分鐘後，採樣檢體，我們發現培養中的KUSA/Al細胞大量繁殖並變為平坦大而多角形（大約20μm×30μm），具有明顯的片狀胞足與樹突狀偽足的類成骨細胞與植體表面及其它細胞互相連結；在這些多形性類成骨細胞間，亦可見零星散佈著小而圓形到紡鍾形的類成骨細胞，另一方面，在免疫組織化學方面，在培養了60分鐘與180分鐘的檢體中發現附著於植體表面的類成骨細胞顯示出肌動蛋白質的存在(F-actin, actin filaments of cytoskeleton)，而穿透性細胞膜接受器蛋白質(α v integrin)(CD51)只在培養了180分鐘檢體中發現。因此，在這次研討中揭示：因不同植體表面誘導衍生的KUSA/AI細胞皆能在不同植體表面緊密附著並大量增生，而其組織學像皆相似。
我們確信在這次的骨引導再生中於培養180分鐘內使得KUSA/A1細胞能在不同表面處理的鈦板上緊密附著，因此我們臆測類成骨細胞能進一步的受控用於骨整合中骨－植體接觸面及遠處基質的骨化作用。

關鍵字

細胞附著 ； 鈦板 ； Kusa/A1細胞 ； 體外實驗

並列摘要

To sustain an extensive osseointegration with optimal bone-to-implant contact (BIC) is one of the essentials for a successful oral implant therapy. In the present in vitro study, we have preliminarily elucidated that KUSA/A1 cells actively proliferated and have differentiated into osteocalcin-producing osteoblast-like cells (Obs) since day 3 of culture. We then cultured the Obs on titanium (Ti) and Ti alloy plates (discs), which have commercially available surface texture and modifications (i.e., anodic oxidization: AO, hydroxyapatite coating: HA, precision blasting: SPI) as same as the current dental implants. The histology of proliferating Obs cultured on discs (Disc-AO, Disc-HA and Disc-SPI; for 180 minutes) was investigated to study the microenvironment mimic initial osseointegration of the dental implant therapy. The present SEM of 60-min Disc-AO, Disc-HA and Disc-SPI specimens demonstrated a loose distribution of spherical-to-ovoid (10 μm in diameter, avg.) Obs migrating towards concavities of the substrate. The proliferating Obs had the bulging cell body sending out many minute filopodia and some lamellipodia to attach with the substrate; few Obs with intercellular junctions were dispersedly found. In contrast, in the 180-min Disc-AO, Disc-HA and Disc-SPI specimens, we observed the cultured KUSA/AI cells actively proliferated and spread into flat, large polygonal (20 μm × 30 μm, approx.) Obs with several prominent lamellipodia and many dendritic filopodia to employ cell-to-disc and intercellular attachments; a few scattered small spherical-to-spindle shaped Obs were also observed interposing the large polymorphic Obs. On the other hand, the present immunohistochemistry of the attached Obs demonstrated the expression of F-actin (actin filaments of the cytoskeleton) in both 60-min and 180-min specimens, and CD51 ( α V integrin) only in 180-min. specimens. Hence, the present study revealed that the condition-induced KUSA/A1 cells actively proliferated and extensively adhered on different substrates yet showed similar histology. We confirmed that the present GBR engineered KUSA/AI cells to obtain extensive cell attachment on different microtextured Ti discs within 180 min, thereby we surmised that the Obs might be further regulated to precipitate mineralization matrix for contact and distant osseointegration.

並列關鍵字

Cell attachment ； Titanium plate ； KUSA/A1 cell ； in vitro