| Abstract: | 骨頭於張力或壓力下會產生重塑反應。牙周韌帶細胞位於牙根周圍與齒槽骨間的結締組織內，承受與分擔牙齒所受到的壓力與張力，於此力學變化下，牙周韌帶細胞進行生長修飾，可生成破骨細胞與成骨細胞，讓牙周組織產生變化。臨床上可能因先天因素或是後天環境影響，導致齒槽骨或是顎骨吸收與破壞，形成骨缺損，因此治療上需讓骨頭再生或是重建，改善牙周疾病或是利用植牙來修復咬合功能等。矽酸鈣材料應用越來越廣泛，材料可以刺激骨細胞增殖，誘導骨生成。現今3D生物列印技術應用廣泛，結合適當之生物墨水，可以依不同形狀製作出三維細胞支架，提供細胞生長。
研究目的：將牙周韌帶細胞添加矽酸鈣之材料，利用生物墨水列印細胞支架，並置於體外張力測試機的拉力下，共同放置於細胞培養箱中，觀察牙周韌帶細胞的骨分化反應。
研究方法：先進行矽酸鈣粉末製備與合成；GelMA明膠製備；製作材料試片；以傅立葉紅外光譜(FTIR)分析成分；進行材料流變分析；試片機械強度分析；進行體外浸泡試驗；進行動態拉伸培養系統測試。之後進行生物相容性測試，觀察細胞存活率，細胞分化路徑與進行細胞骨化分化能力。
研究結果：牙周韌帶細胞添加於GelMA明膠矽酸鈣之材料進行3D生物列印出細胞支架，在培養箱中以張力拉力機施予支架力量。細胞支架之強度隨著矽酸鈣濃度增加而增加（具統計學上差異，p<0.05）。支架之成分於XRD/FTIR分析下顯示支架並無因列印後而改變其化學成分。細胞可以正常生存於培養箱中，且含有矽酸鈣的細胞生長較好（具統計學上差異，p<0.05）。施予張力下測試細胞之存活率，含有矽酸鈣之細胞的FAK蛋白表現較對照組高（具統計學上差異，p<0.05）。細胞之吸附效果上，F-actin蛋白表現，含有矽酸鈣之細胞表現量較對照組高（具統計學上差異，p<0.05）。牙周韌帶細胞於本試驗的分化乃經ERK磷酸化進行細胞傳遞，且含有矽酸鈣之細胞表現較強（具統計學上差異，p<0.05）。在牙周韌帶細胞骨分化標記蛋白ALP，OPN和OC表現的檢測也呈現含有高濃度矽酸鈣支架之細胞ALP，OPN和OC蛋白表現較強（具統計學上差異，p<0.05）。
結論：牙周韌帶細胞添加於3D生物列印出含明膠矽酸鈣之細胞支架，其結構符合材料應有之強度與降解率；於具有模擬細胞張力下之環境一起培養，施以張力拉力的力量，在培養後的牙周韌帶細胞具存活率佳，有細胞相容性；牙周韌帶細胞可以進行骨分化反應。期望日後可以藉由本文研究此模式來改善骨缺損等問題．
In bone biology, different kinds of force may result in various biological bone change. In general, the bone will be resorbed when the force comes from a pressure site and the it will be deposed when the force is from a tension site. In oral, periodontal ligament cell is located in the root and alveolar space which contains connective tissue, vessel, periodontal ligament cells, nerve and etc. Under the force, the periodontal tissue will reach a balance, and the bone structure will be change simultaneously. Clinically, the alveolar bone or jaw bone defects may arise from the genetic factor or environment factors which lead to alveolar bone resorption or destruction. The clinical dental treatment is focused on bone rebuilding or regeneration. If succeed, the periodontal disease can be improved and the implant therapy is feasible. The calcium silicate material has been used in dental treatment for many years. From past studies, this material can induce the cell proliferation and osteogenesis. On the other hand, due to the improvement in 3D bioprinting technique with the help of proper bioink, the cell can be printed by a 3D bioprinting to fit in any shape of bony defects.
The purpose of this study was to evaluated the ossifying reaction of a 3D bioprinting periodontal ligament cells, under the tension condition.
Material and methods:
By combining the 3D bioprinting methods, a 3D-form fabricated PDL cell scaffold was placed in an incubator to co-cultured it with a cyclic tension test machine. The first step is to produce calcium silicate powder. We set up a GelMA gelatin and placed it on a sheet for testing. We also used the XRD, FTIR to analyze the scaffold compositions. A reference data for the tensile strength machine was set to co-culture with the PDL implemented calcium silicate cytoskeleton so that the biocompatibility assay can be performed. The PDL differentiation pathway was done by the ERKinase expression, and the Alizarin stain assay is applied to detect the PDL ossifying effects.
Results:
The PDL cell that contained a 3D bioprinting scaffold showed a better result in the strength and degradation test than the control group, which is the one without the calcium silicate materials, during the mechanical test. The 3D bioprinting didn’t change the composition and chemical structure. The PDL proliferation was higher on CS1 group, and both CS1, CS0.5 groups had statistical higher proliferation rate than the control group (P<0.05). The FAK protein expression was highest on CS1 group (P<0.05). The F-Actin was higher on CS1 and CS 0.5 groups (P<0.05), representing the PDL attachment was doing well. The PDL differentiation pathway was from the pERKinase pathway. The CS1 group showed a highest pERK expression (P<0.05). The ossifying ability test showed the CS0.5 and CS1 groups were statistical higher than control group(P<0.05).
Conclusion:
The PDL cell implemented in calcium silicate 3D bio-printing cytoskeleton can have good physical properties without changing its original chemical characteristics. The good cell proliferation and ossifying ability of this scaffold could be applied to improve the bony defect regeneration in the future. |
