幹細胞的研究中，如何萃取與分離幹細胞是重要的部分。成體幹細胞可分為有血液幹細胞與間葉幹細胞。從過去的研究顯示，間葉幹細胞可以分化為造骨細胞、造軟骨細胞、脂肪細胞，甚至可以分化為心肌細胞或神經細胞。在間葉幹細胞的來源中，主要是自成人的骨髓。但在口腔中，有學者從脫落的乳牙牙胚、牙根周圍的牙周膜、發育中的根尖組織均分離出間葉幹細胞，並證明其分化能力。在牙周病發展的過程中，會有發炎細胞的浸潤，進而各種的發炎物質刺激下，形成牙周發炎組織，破壞齒槽骨。通常在牙周手術過程中，我們會將肉芽組織移除丟棄。假設可以從肉芽組織分離出間葉幹細胞，進而運用在牙周再生治療中。將是很有意義的，而且不用擔心免疫排斥的問題。
首先我們利用牙周手術取出的發炎組織，用免疫染色，以間葉幹細胞表面抗原(STRO-1)標定，確認了發炎組織中的確有間葉幹細胞的存在。接下來將發炎組織的細胞分離，在培養皿中進行細胞培養。用低密度細胞群聚的模式，讓單一細胞形成一個一個群聚。再運用細胞染色的方式，將有表現間葉幹細胞表面抗原的細胞株加以培養。等培養到一定量，運用不同的培養條件，讓細胞株進行分化。分化形成成骨細胞、軟骨細胞、脂肪細胞。並運用不同的細胞標記抗體，驗證所分化的細胞的確表現骨細胞、軟骨細胞及脂肪細胞的特徵。接下來利用動物實驗模型驗證，用免疫缺陷的小鼠，在頭蓋骨製造關鍵大小的骨缺陷，用肉芽組織的幹細胞，運用在小鼠頭蓋骨缺損的修復，等術後八周，比較結果。發現在實驗組及對照組之間，的確因著肉芽組織的幹細胞存在與否，骨缺損的修復有著差異。
這代表著肉芽組織分離的間葉幹細胞，具有分化成不同細胞的能力，也有促進骨缺損的修復。希望以後可以藉由自體間葉幹細胞的移植，進而增進牙周治療的再生效果。

Isolating the stem cells from tissue is an important part for the research. There are two difference sources, the embryonic stem cells (ESCs) and the post-natal stem cells. The post-natal stem cells can be defined as the hematopoietic stem cells (HSCs) and the mesenchymal stem cells (MSCs). The MSCs can differentiate to the osteoblast, the chondrocyte, the adipocyte, and even the cardiac cells or neural cells. Most of the reports isolate MSCs from the bone marrow, but there are still some other sources of the MSCs. Regarding the oral tissues, MSCs have been isolated from the exfoliated deciduous teeth, from the periodontal ligament tissue, and from the dental pulp. These cells have the MSCs cell markers and can differentiate into osteoblast, chondrocyte, and adipocyte. In the process of periodontal disease, there are many inflammation cells infiltration. These cells secrete cytokines and irritate the granulation tissue formation. This procedure can lead to the alveolar bone resorption. In the periodontal surgery, we usually remove the granulation tissue, and graft the periodontal defect. It is meaningful to isolate the MSCs from the granulation tissue and use these cells in the following regeneration therapy. Rejection is not a concern from these cells.
In this research, we first took the granulation tissue from periodontal defects, and used immunohistochemistry (IHC) to mark the MSCs. We used the STRO-1 as primary antibody and found the MSCs in the granulation tissue. Then we isolated the cells from the granulation tissue and used the CFU-F cell culture method. Then we isolated the STRO-1 positive cell clones. We used different conditions to induce these cells to differentiate into osteoblast, chondrocyte and adipocyte. We further confirmed the result by cell markers (the osteopontin, collagen II, PPAR-γ) and special staining assays (the Alizarin red, the Alcian blue and Oil-red staining). In the following animal experiments, we used the immunocompromised mice model, create a critical sized defect at calvaria (3 mm), and compare the result for test and control groups after 8 weeks. After examining the cranial specimens from test and control groups, the test group had significantly greater bony deposition area than control group.
The granulation tissue derived stem cells can differentiate into osteoblast, chondrocyte and adipocyte, and can improve the repair of the bony defect. We propose the granulation tissue derived stem cells may improve the result of the periodontal surgery in the future.

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