生醫材料培養人類牙髓與牙周韌帶多細胞球體之生物特性與應用潛力探討

Abstract

近年來，幹細胞在組織修復再生治療等各種適應症的應用已被廣泛探討及研究。間質幹細胞（Mesenchymal stem cells ，MSCs）具有自我更新能力和分化潛能，MSCs可以從成體組織分離取得，例如骨髓、脂肪組織、外周血、以及胎盤、臍帶血和臍帶等圍產期組織，此外，齒源組織也能分離出MSCs供作幹細胞研究及應用，例如牙齦、牙根尖乳突、牙周韌帶、牙髓等不同部位都蘊藏有MSCs。許多研究陸續顯示相較於傳統2D平面細胞培養，多細胞球體的3D培養環境更有利於MSCs維持細胞活性及分化潛能。 本研究以生醫材料促成人類牙齒幹細胞聚集成多細胞球體，分析成球細胞的增生、遷移、及分化能力，並檢測成球細胞的特定蛋白表現及相關訊息路徑，更進一步探討應用多細胞球體培養於3D組織工程支架是否能促進傷口癒合。我們取人類智齒培養牙髓細胞及牙周韌帶細胞進行實驗，二種細胞皆先以流式細胞儀分析確認CD44、CD73、CD105和STRO-1等幹細胞表面抗原表現。將牙髓細胞培養於塗佈 chitosan (CS) 或polyvinyl alcohol (PVA)的培養盤，細胞會逐漸聚集成多細胞球體，在培養條件最佳化之後，可發現細胞球體大量表現缺氧因子（Hypoxia-inducible factors-1 alpha, HIF-1α），vascular endothelial growth factor (VEGF)表現也隨之提升，透過cell migration分析，從3D多細胞球體向外生長的細胞，相較於培養於2D平面的細胞，表現出較好的遷移能力。細胞成球過程中加入YC-1 (HIF-1α inhibitor)，VEGF的表現會被抑制。此外，Y-27632 (Rho inhibitor)會完全抑制chitosan膜上牙髓細胞的聚集和多細胞球體形成，而對PVA膜上的細胞成球現象則不會有抑制作用。Rho A的訊息通路參與牙髓細胞在chitosan膜上聚集和形成多細胞球體形成的過程。我們進一步將培養於chitosan的牙髓多細胞球體培養於3D列印的聚乳酸支架中，再施以骨分化誘導，結果發現鹼性磷酸酶表現有所增加，而且細胞外基質礦化程度上升，顯示透過多細胞聚集成球體的過程可促進牙髓細胞在體外環境培養中成骨/成牙本質的分化反應。 如同牙髓細胞的反應，培養於CS或PVA的牙周韌帶細胞也會自動聚集成多細胞球體，細胞成球刺激下HIF-1α表現明顯增加。透過體外實驗也顯示牙周韌帶細胞聚集為多細胞球體仍可維持其活性，細胞從球體移行爬出的遷移能力明顯被提升。此外，我們發現使用適當濃度的EDTA可維持細胞活性並弱化細胞成球現象，而且前述多細胞球體促進細胞遷移能力表現的效應更為明顯。將牙周韌帶細胞球體種入3D列印的膠原蛋白組織工程支架中，以糖尿病鼠動物實驗模式探討3D支架含成球細胞對於皮膚傷口癒合能力的影響，實驗結果顯示chitosan薄膜上培養的牙周韌帶多細胞球體具有促進皮膚傷口組織修復的能力。 上述研究結果顯明: 應用適當生醫材料能促成人類牙髓細胞與牙周韌帶細胞自動聚集成為多細胞球體，被移置回種於常規培養環境後，成球細胞表現較高的遷移能力。將牙髓多細胞球體載入聚乳酸支架繼續培養，相較於培養於2D平面的對照組細胞，3D成球細胞具有好的體外成骨分化能力。將牙周韌帶多細胞球體載入膠原蛋白3D支架培養，透過小鼠實驗觀察生物體內反應，則驗證膠原蛋白支架承載牙周幹細胞球體能夠顯著促進皮膚傷口癒合。與此同時，我們期許研究成果未來能夠應用於組織工程再生領域。

In recent years, the clinical application of stem cell therapy has been widely investigated in tissue engineering and regenerative medicine. Mesenchymal stem cells (MSCs) are capable of self-renewal and multi-lineage differentiation. MSCs can be isolated from somatic tissue, e.g. bone marrow, adipose tissue, peripheral blood cells, and perinatal tissue including placenta, umbilical cord blood, as well as umbilical cord. MSCs can also be obtained from dental tissue, such as gingiva, apical papilla, periodontal ligament and pulp, which are useful in stem cell research and therapeutic application. Previous studies revealed that MSCs multicellular spheroids cultured within 3D environment is beneficial to the maintenance of cell viability and differentiation potency compared to conventional 2D cultures. In present study, we analyzed the proliferation, migration and differentiation abilities of spheroid cells and determined specific proteins and relevant signaling pathways via biomaterial-induced spheroid formation of human dental stem cells. We also discussed if multicellular spheroids cultivated in 3D tissue engineered scaffolds can promote wound healing. Human dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) were used in present study and the stem cell surface marker expressions such as CD44, CD73, CD105 and STRO-1 were determined by flow cytometry in both cells. The pulp cells were applied over chitosan (CS) or polyvinyl alcohol (PVA) dish where the cells gradually aggregated into multicellular spheroids. We found that optimizing the culture condition leads to high expression of hypoxia-inducible factors-1 alpha (HIF-1α) as well as an increase of vascular endothelial growth factor (VEGF) expression in cell spheroids. Cells migrated outward from 3D spheroids exhibited better migration ability compared to 2D cultured cells as analyzed by transwell cell migration assay. VEGF expression was inhibited by adding YC-1 (HIF-1α inhibitor) in the process of spheroid formation. Besides, DPCs aggregation and multicellular spheroid formation on chitosan membrane were completely blocked with the treatment of Y-27632 (Rho inhibitor) but such inhibitory effect did not occur in PVA membrane. Rho A signaling pathway participates the process of dental pulp cell aggregation and spheroid formation on chitosan membrane. We transferred multicellular pulp spheroids onto 3D printed polylactic acid scaffolds and performed induction of osteogenic differentiation. As a result, the expression of alkaline phosphatase elevated and extracellular matrix mineralization was enhanced, which is evident that the aggregation of multicellular spheroids promotes in vitro differentiation of osteogenesis/odontin of pulp cells. Similar to the response of dental pulp cells, PDLCs cultivated in CS or PVA spontaneously gathered into multicellular spheroids and the formation of spheroids stimulated the increase of HIF-1α expression. In vitro wound healing test showed that the multicellular spheroids formed by PDLCs are viable and the ability of cell migration has been improved obviously. Furthermore, we found that treatment with EDTA at appropriate concentration might maintain cell viability and diminish spheroid formation. EDTA conditioning also contributes to an obvious increase of cell migration ability. We studied the effect of 3D scaffolds culturing spheroids towards cutaneous wound healing ability in diabetic mice by seeding PDLC spheroids onto 3D printed collagen scaffolds and the outcome revealed that 3D collagen scaffolds culturing PDLC spheroids possess the ability to promote wound healing. From these results, it is clear that self-aggregated DPCs and PDLCs spheroids of which formation was induced by appropriate biomaterial being recultivated in a normal culture environment exhibit greater migration ability. The 3D multicellular spheroids developed from DPCs, if further cultivated in polylactic acid scaffolds, portray superior abilities of in vitro osteogenic differentiation as compared to 2D (TCPS) cultivated cells. On the other hand, the diabetic mice experiment involving the multicellular spheroids of PDLCs which were transferred and being cultivated in 3D collagen scaffolds to determine in vivo biological response proves that collagen scaffolds carrying periodontal stem cell spheroids can promote cutaneous wound healing obviously. Henceforth, the outcome of present study is expected to be practicable in the field of tissue engineering and regenerative medicine in future.