Gelatin/Chondroitin-6-Sulfate/Hyaluronan Tri-copolymer Scaffold and dental dental bud cells for Swine Teeth Regeneration

牙周疾病、齲齒、撞傷而造成牙齒缺損，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 個月齡迷你豬，將未萌發之臼齒以外科手術取下，其後分離牙胚組織切碎成＜1 mm3的小片段，於培養液中以37℃，5 ％ CO2培養。將培養的牙胚細胞 (Dental bud cells; DBCs) 種入明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架 (Gelatin/chondoitin-6-sulfate /hyaluronan tri-copolymer scaffold; GCHT scaffold) 內培養一週。實驗組將含有DBCs/GCHT scaffold植入同源豬原齒槽骨內及下顎骨骨質中，並取部份DBCs/GCHT scaffold以10 ％中性福馬林及2.5 ％戊二醛固定分別製作組織切片、進行環境掃描式電子顯微鏡掃描，可見細胞於支架中貼附生長並形成細胞外基質。植入24及36週後以X光放射線攝影評估牙齒生長情形，並於36週犧牲豬隻以組織切片評估；對照組為取其臼齒後不種入DBCs/GCHT scaffold者3頭，及取臼齒組織後僅種入不含細胞的支架之豬隻3頭。實驗組6頭豬隻植入DBCs/GCHT scaffold，結果在3頭豬隻原齒槽骨出現放射不透明區域，而另3頭在X光放射線攝影則無任何發現。3頭在原齒槽骨出現放射不透明區域的豬隻，其中2頭各別可見約1×0.5 ㎝2和 0.5×0.5 ㎝2牙齒鈣化形態；另1頭則是出現直徑1 ㎝的放射線不透明區域。再生的牙齒進行組織學的分析，在蘇木紫及伊紅染色 (Haematoxylin and Eosin stain; H&E )，2頭中可觀察到Dentin/pulp-like complex之牙齒結構出現；免疫組織化學染色則可證實牙齒質具有Bone sialoprotein的表現。另1頭圓形的放射線不透明區域則在H&E染色下見到骨組織出現。由上述的結果再次證實，DBCs具有自行更新、分化成不同後裔及選植生殖 (Clonogenic) 的能力。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組織再生，也證實牙齒幹細胞可能存於牙髓組織中。

Tooth loss due to periodontal disease, dental caries and trauma affect humans and animals adversely at some time in their lives. A biologically regenerated tooth could replace a lost tooth and the new substituted tooth would provide a vital alternative to currently available clinical treatments. For this purpose, we used a tissue engineering approach to research tooth regeneration. Using surgical operation to remove a molar tooth from a 1.5 month-old miniature pig, before eruption, and then minced dental bud tissues into < 1 mm3 pieces to culture in medium at 37℃ with 5 ％ CO2 . The dental bud cells (DBCs) were isolated, re-suspended, and then the cell suspension was injected into Gelatin/chondoitin-6-sulfate /hyaluronan tri-copolymer scaffold (GCHT scaffold). The DBCs/GCHT scaffold was cultured in a spinner flask bioreactor for 1 week. The DBCs/GCHT scaffold structures were implanted into the pig's original alveolus. In addition, a small piece of DBCs/GCHT scaffold sample was individually fixed with 10 ％ neutral formalin and 2.5 ％ glutaraldehyde. The results showed DBCs growing and attaching on GCHT scaffold, and even secreted extracellular matrix. In experimental groups: 6 pigs were implanted with DBCs/GCHT scaffold into the original alveolus. After 36 weeks, regenerated teeth could be found in 3 pigs by X-ray examination, especially a recognizable 1×0.5 ㎝2 and 0.5×0.5 ㎝2 size tooth formed in 2 different swine mandibular alveolus, and in other swine can see a 1 cm diameter radio-opaque region, the other 3 pigs had no notable results. Which 2 pigs showed tooth regeneration containing dentin/pulp-like complex tooth structures which appeared by H&E staining. We also detected bone sialoprotein (BSP) expressed in dental pulp, odontoblast and dentin by immunohistochemical (IHC) methods. A 1 cm radio-opaque region in a pig stained by H&E staining only presented bone tissue. Above all, we demonstrate the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of DBCs. In control groups: 3 pigs molar tooth was removed but no implanted DBCs/ GCHT scaffold and 3 pigs receival merely implanted GCHT scaffold. After 36 weeks, no regenerated tooth was found in the original site. Our results demonstrated regeneration of tooth tissue structures could be achieved by applying tissue engineering techniques and suggested that dental stem cells may appear in dental pulp tissues.