臺灣博碩士論文加值系統

[研究目標] 人類牙周韌帶細胞 (periodontal ligament cells ; PDLCs) 細胞再生相關探討。
[材料方法] 以細胞分枝法(outgrowth)或酶切培養法 (enzyme-digested) 進行體外培養人類牙根中段之牙周韌帶細胞，評估以冷凍保存劑dimethyl sulfoxide (DMSO) 進行冷凍保存及解凍後對細胞活性之影響，及以ethylenediaminetetraacetic acid (EDTA) 進行牙根表面處理後，牙周韌帶細胞再植於牙根的成功率。
[結果討論] 研究結果顯示outgrowth培養法比enzyme-digested 培養法有較高成功率，可能因為減少化學及器械在操作過程中的傷害所致。使用 7.5% 及10% DMSO進行冷凍保存與解凍，對細胞活性並無明顯影響。同時以24% EDTA 進行牙根表面處理後，增加牙周韌帶細胞再植於牙根的成功率，可能因移除牙根表面smear layer後，增加細胞初期在牙根表面附著能力之結果。
[研究結論] 人類牙周韌帶以outgrowth 培養法增殖後，再植於以EDTA表面處理後的牙根，可能增加牙齒自體移植時牙根的再利用率而達成保存醫學的目標。

[Objective] The activity of cell regeneration in human periodontal ligament cells (PDLCs) was characterized.
[Materials and methods] The human PDLCs were isolated from middle third of the tooth root and cultured by using outgrowth or enzyme-digested methods. The cell viability of the PDLCs after cryopreservation with dimethyl sulfoxide (DMSO) was evaluated. The success rate of re-implanted PDLCs onto the root after treatment with ethylenediaminetetraacetic acid (EDTA) was also determined.
[Results and Discussion] Our data indicated that outgrowth culture represented a higher success rate than enzyme-digested culture of PDLCs. It might be due to reducing the damage from chemical and mechanical manipulation processes. There is no difference between cryopreservation with 7.5% and 10% DMSO after thawing of PDLCs. Additionally, roots conditioned with 24% EDTA enhanced the re-implantation of PDLCs onto the root. This may be the reason of removing the smear layer from periodontal root surface and therefore increases the PDLCs adherence on root surface.
[Conclusion] Outgrowth culture expansion and re-implantation of PDLCs onto the autotransplantation root after root conditioning in EDTA might improve the re-utilization of the tooth.

Index
中文摘要 III
Abstract IV
Index V
Table index VII
Figure index VIII
I. Introduction 1
1-1. Introduction of conventional dental treatments 2
1-2. Introduction of dental implants 3
1-3. Background introduction of autotransplantation 4
1-4. The characters and biological functions of PDLCs 6
1-5. Culture methods of PDLCs 7
1-6. Cryopreservation of PDLCs 8
1-7. Magnet cryopreservation of PDLCs 9
1-8. Motivation and purpose 10
II. Materials and methods 11
2-1. Patient selection and clinical data 12
2-2. Definition of the tooth root with periodontal disease 13
2-3. Preparation of human PDLCs 14
2-4. Modified outgrowth culture of hPDLCs with whole root 15
2-5. Modified enzyme-digestion method of hPDLCs 16
2-6. Cryopreservation of PDLCs-DMSO 17
2-7. Immunofluorescence staining-CD90, CD105 18
2-8. Cell differtiation assay-ARS staining 19
2-9. Cell adhesion assay-crystal violet staining 20
2-10. Cell viability assay-MTT assay 21
2-11. Cytotoxicity assay-LDH assay 23
III. Results 24
3-1. Isolation of PDL cells–outgrowth and enzyme-digested cultures 24
3-2. Cryopreservation and thaw of PDL cells 25
3-3. Expression of stem cell markers on PDL cells 26
3-4. Cell differtiation assay-ARS staining 27
3-5. Re-implantation of PDL cells onto the root 28
3-6. Cell viability of PDL cells treated with EDTA 29
IV. Discussion 30
4-1. Comparison of outgrowth and enzyme-digested methods 30
4-2. Modified culture method of hPDLCs with whole root 32
4-3. Cryopreservation and thaw of PDL cells 33
4-4. Re-implantation of PDLCs onto the root 34
4-5. Expression of stem cell markers on PDLCs 35
4-6. Limitation and future works 36
V. Conclusion 37
References 38

Table Index

Table 1. Comparison of survival and success rates of removable partial dentures (RPDs), fixed partial dentures (FPDs) and implants. 42
Table 2. Comparison of survival and success rates of autotransplantations (ATs) 43
Table 3. Comparison of PDLCs primary culture with outgrowth (OG) and enzymatic digested (ED) methods 44
Table 4. Primary culture of hPDLCs with outgrowth (OG) and enzyme-digested (ED) methods in periodontitis teeth (P) and normal teeth (N) 45
Table 5. Cell viability test and controlled group of MTT assay in periodontitis teeth (P) and normal teeth (N) with 24% EDTA root conditioner 46
Table 6. Results of hPDLCs primary culture with outgrowth (OG) and enzyme-digested (ED) methods in periodontitis teeth (P) and normal teeth (N). 47

Figure Index

Figure 1. PDLCs outgrowth culture and re-implantation. 48
Figure 2. Cell viability test and controlled group of MTT assay in periodontitis teeth (Peri) and normal teeth with 24% EDTA root conditioner. 49
Figure 3. Microscopic observation of human periodontal ligament (hPDL) primary cells and human gingival (hGF) primary cells. 50
Figure 4. Success rate of hPDLCs with various primary culture 51
Figure 5. Cell viability test of MTT assay with various primary culture methods in periodontitis teeth (perio). 52
Figure 6. Cryopreservation and thaw of PDLCs. 53
Figure 7. Expression of stem cell markers on PDLCs. 54
Figure 8. Alizarin red S (ARS) staining of cell mineralization in PDLCs and HGF. 55
Figure 9. Crystal violet staining of PDL cells on tooth root which was treated with 24% EDTA.. 56
Figure 10. Cell viability of PDLCs treated with EDTA. 57
Figure 11. Normal and perio PDLCs re-implantation on treated root 59

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