行政院國家科學委員會專題研究計畫成果報告:明膠軟骨透明質酸三重聚合物支架進行齒芽組織工程

牙周疾病、齲齒、撞傷或基因失調而造成牙齒缺損，一直是許多人和動物生 活中極大的困擾，若能發展出生物性的牙齒再生取代缺損的牙齒將是臨床治療的 一種新方法。本研究之目的乃是利用組織工程的方法達成牙齒的再生。取1.5 個 月齡迷你猪，將未萌發臼齒以外科手術取下，放入30 m1 含1.5 % PC/ST (Penicillin /streptomycin)之DMEM (Dulbecco´s modified eagle medium)中於4℃保存。其後分 離臼齒中的牙胚組織，以1X PBS（Phosphate buffer saline）液清洗乾淨後切碎成 ＜1 mm3的小片段，於含1.5 % PC/ST及20 % FCS之DMEM中37℃,5 ％ CO2培 養。培養3 星期使細胞數達1×107個。將培養的齒芽細胞種入明膠-軟骨素-透明 質酸三重聚合物支架內培養一週。實驗組將含有細胞/支架埋入同源猪原齒槽骨 內及下顎骨骨質中;兔子則埋入末端肋軟骨之皮下，並取部分細胞/支架以10 ％ 中性福馬林及2.5 ％戊二醛固定分別製作組織切片、進行環境掃瞄式電子顯微鏡 掃瞄，可見細胞於支架中的生長情形。植入24 及36 週以X光放射線照影評估牙 齒生長情形，並於36 週犧牲猪隻及兔子以組織切片評估;對照組為取其臼齒後 不種入細胞/支架者6 隻，及取臼齒組織後僅中入不含細胞的支架之猪隻3 頭。6 頭植入細胞/支架的猪隻，3 頭在原齒槽骨出現放射不透明區域，而3 頭在X光放 射線照影則無任何發現。3 頭在原齒槽骨出現放射不透明區域的猪隻，其中一頭 可見約1×0.5×0.5 ㎝3牙齒萌發於口腔外;一頭則在齒槽骨內可見牙齒鈣化型態; 另一頭則是出現直徑1 ㎝的放射線不透明區域。萌發的牙齒進行H&E染色可見 Dentin/pulp-like complex之牙齒相關結構出現，而Goldner's及免疫化學等染色則 在進行中。兔子再生猪牙齒試驗上則無任何發現。對照組取牙齒組織相對位置都 無牙齒生長。本研究證實可由組織工程的方法達成牙齒再生，也證實牙齒幹細胞 存於牙髓組織中。Tooth lost due to periodontal disease, dental caries, trauma, or varieties of genetic disorders continuing to affect human and animals adversely at some time in their lives. A biological regenerated tooth could replace lost tooth, the new substituted tooth would provide a vital alter-native to currently available clinical treatments. For this purpose, we used a tissue engineering approach to research the tooth regeneration. Using surgical operation to remove 1.5 month-old miniature pig molar tooth before eruption and placed into 30 mL of DMEM (Dulbecco´s modified eagle medium)+1.5 ％ PC /ST (Penicillin/streptomycin) at 4°C. Dental bud tissues were washed by 1X in PBS（Phosphate buffer saline）, minced into < 1 mm3 pieces, and then cultured in DMEM +1.5 ％ PC /ST + 20 % FCS at 37℃ with 5 ％ CO2. After 3 weeks cell yielded 1 x 107 cells, and seeded cells onto Gelatin-Chondroitin-Hyaluronan, Tri-copolymer scaffold. Cultured for one week, dental stem cells were migrated into and attached the scaffold. The cell/scaffold constructs were implanted in both autograft into pig and xenograft into rabbit. In addition, a piece of little cell/scaffold sample was fixed individually with 10 ％ neutral formalin and 2.5 ％ glutaraldehyde, and then growing cells were demonstrated in scaffold by histological section analysis and Environmental Scanning Electron Microscope (ESEM) individually. In experimental groups: 6 pigs were implanted with cells/scaffold (C/S) into original alveolus, rabbits were implanted with C/S into subcutaneous tissue near the end costal cartilage. After growing in pigs and rabbits for 24 and 36 weeks, There were regenerated growing teeth could found in 3 pigs by X ray examination, especially a recognizable 1×0.5×0.5 ㎝3 size tooth formed in swine alveolus bone by evaluated with radiography, but the other 3 pigs were no found. In rabbit we did not saw anything-like tooth in original implanted place. There was a tooth containing dentin/pulp-like complex tooth structure was appeared by hematoxylin and eosin staining in histological analysis. In control groups: removed molar tooth but did not implanted cell/scaffold 6 pigs group and merely implanted scaffold 3 pigs group. After growing for 24 and 36 weeks in pigs, no any regenerated tooth was grown in original place. Our results demonstrated regeneration of tooth structures could achieved by apply tissue engineering technique and suggested that dental stem cells appearance in dental pulp tissues