明膠軟骨素透明質酸三重聚合物支架配合牙胚細胞併用下顎骨骨髓幹細胞移植之組織工程

Abstract

牙齒經常因牙周疾病、齲齒、撞傷或癌症而造成缺損，如果能發展出生物性的牙齒再生以取代缺損的牙齒將是臨床治療的一種新療法。本研究之目的乃是利用組織工程的方法達成整顆牙齒的再生。骨髓中的骨髓間葉幹細胞 (Bone marrow mesenchymal stem cell；BMSC) 為一具有多分化能力的幹細胞，在BMSC分化成骨組織的骨化過程 (Osteogenesis) 中，BMSC可分泌許多生長因子 (Growth factor) 和形態原 (Morphogens)，包括了Bone morphogenetic protein (BMP)、Transforming growth factor-beta (TGF-beta) 、Fibroblast growth factor (FGF) 、Platelet-derived growth factor (PDGF) 及Insulin-like growth (IGF) 等，這些生長因子及形態原與牙齒胚胎發育及生長所需的生長因子與形態原有部份是相同的，故本實驗試著將骨髓導入生牙作用以期BMSC可協同牙胚細胞和支架促進牙齒的再生。取1.5月齡迷你豬的未萌發牙胚組織，切碎成＜1 mm3的小片段，於DMEM培養液中加入20 ％胎牛血清，以37℃，5 ％ CO2培養，待細胞量逹到1×106 cell/ml後，將牙胚細胞全數打下並種入明膠/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，異種移植至裸鼠後背部皮下處。於4週、8週、12週、16週、20週及24週後分次犠牲，以H&E、VonKossa和免疫組織化學染色評估牙齒再生部位。豬隻方面，則由豬隻原齒槽骨內抽取骨髓混合牙胚細胞，再全數種入 GCHT scaffold內且靜置30分鐘，再將牙胚細胞/骨髓/支架自體移植至豬隻原齒槽骨內，待40週後以X光放射線攝影、H&E、VonKossa和免疫組織化學染色評估牙齒生長情形。裸鼠結果顯示，於4週、8週和12週可見到 Cytokeratin 14 (CK14) 的表現，12週還可見 Vascular endothelial growth factor (VEGF)表現，16週、20週和24週則可見到類牙本質物質形成且表現 Dentin matrix protein-1 (DMP-1)，另外於24週還可見到骨細胞和骨組織的生長並表現出 Osteopontin (OPN)。而9隻實驗豬中，9隻皆可在X光放射線攝影下見到放射線不透明區域的出現。其中1隻於H&E切片下可到牙本質的生成且在免疫組織化學染色下表現出DMP-1；另1隻則可見到再生牙齒萌發於口腔，於H&E切片染色下可觀察到齒冠、齒根、牙髓腔、牙釉質、牙釉質母細胞、牙本質、牙本質母細胞、牙本質小管、牙骨質、血管、牙周韌帶和齒槽骨的生成；而在免疫組織化學染色結果指出，牙本質、前牙本質、牙本質母細胞和牙本質小管皆表現 DMP-1；齒槽骨和牙骨質可見到 OPN蛋白表現；牙髓腔內之血管內皮也表現出VEGF蛋白。本實驗證實了利用組織工程配合適當支架併用豬牙胚細胞和骨髓間葉幹細胞，在裸鼠異體移植與豬隻自體移植狀態下，可完成牙齒再生；並證實了下顎骨骨髓幹細胞可幫助牙齒相關組織的生成。

Tooth loss due to periodontal disease, caries of the teeth, trauma and oral cancers to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. The purpose of this project is to generate a complete tooth crown by tissue engineering. Bone marrow mesenchymal stem cells (BMSC) are multipotent stem cells capable of differentiating into various cell types which exist in bone marrow. BMSC can differentate to osteocyte when bone growth during the osteogenesis. There are a lot of growth factors and morphogens in osteogenesis, including bone morphogenetic protein (BMP)、transforming growth factor-beta (TGF-beta)、fibroblast growth factor (FGF)、platelet-derived growth factor (PDGF) and insulin-like growth (IGF). Partial of those growth factors and morphogens which involve in tooth development are match with osteogenesis. The aim of this project is to combined the dental bud cells (DBCs)、BMSC and gelatin/chondoitin-6-sulfate /hyaluronan tri-copolymer scaffold (GCHT scaffold) to regenerate a complete tooth. Removed the third 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 DMEM medium with 20 ％ fetal cow serum at 37℃ with 5 ％ CO2 . When the DBCs yield reached 1×106, the DBCs were isolated, re-suspended, and then the cell suspension was injected into GCHT scaffold. The DBCs/GCHT scaffold constructs were implanted in xenograft into nude mice’s dorsum. After xenogreat implantation for 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks and 24 weeks, there were evaluated by hematoxylin and eosin stain, VonKossa stain and immunohistochemical (IHC). The results of nude mice showed 4 weeks, 8 weeks and 12 weeks expressed Cytokeratin 14 (CK14) protein and even expressed Vascular endothelial growth factor (VEGF) in 12 weeks. In 16 weeks, 20 weeks and 24 weeks generated dentin like structures and expressed Dentin matrix protein-1 (DMP-1). 24 weeks generated osteocyte and bone tissues which expressed Osteopontin (OPN). In the swine experimental groups, the radiographic analyses were performed for 9 pigs, the results showed some opaque areas were formed in 9 pigs. One of the 9 pigs observed dentin like structures and expressed DMP-1 and the other one pig generated whole crown, root, pulp, enamel, ameloblast, dentin, odontoblast, dentin tubular, cementum, blood vessel, periodontal ligament and alveolar bone. The immunohistochemical analysis results showed the dentin, dentin tubular and odontoblast expressed DMP-1, cementum and alveolar bone expressed OPN and the vascular endothelial cell of pulp expressed VEGF, too. We demonstrated that the porcine dental bud cells combined with bone marrow mesenchymal stem cells and suitable scaffold can regenerated tooth by tissue engineering when porcine model with autogenic cell transplantation and nude mice model with xenogenic cell transplantation. We also demonstrated the bone marrow mesenchymal stem cells could promote the structures of tooth regeneration.