臺灣博碩士論文加值系統

組織工程使用支架、細胞以及影響因子等三種要素，來再生器官以及組織。自體移植骨，因具有支架、細胞、以及骨形成蛋白質的特性，是最佳的骨移植物。然而，自體移植骨取得不易、須延長手術時間，更造成手術取移植骨的捐贈區位置不適，都限制了自體移植骨的應用。因此，應用組織工程技術，作為治療骨缺損的另一種選擇，用以取代來源稀少的自體移植骨。
第一型膠原蛋白是骨骼內含量最多的細胞外基質蛋白質。膠原蛋白支架因具有孔洞、良好的生物相容性、親水性、降解能力、以及具有增進細胞吸附、增生與分化的特性，因此被大量的應用於骨工程學。骨細胞在支架內需經過增生、分化以及鈣化的過程，最終才能形成骨骼。
交聯處理以及膠原蛋白支架形成過程中的溫度控制，是製造膠原蛋白支架的重要控制因素。交聯處理可以增加膠原蛋白支架的機械強度並減緩降解速率；而溫度控制，則可控制孔洞形狀與大小。本實驗的主要目的是檢測不同條件下形成的膠原蛋白支架，其體外與體內的測試反應。
膠原蛋白來源，是從八週齡大鼠尾巴，利用重複的酸溶解以及鹽析，並經由酵素（pepsin）剪切尾端，以純化出第一型膠原蛋白。利用溫度，重組以及交聯作用來控制支架的結構以及機械強度。發現在中性溶液，37°C條件下進行重組，膠原蛋白會成為不透明的膠狀物。但這種現象為可逆性，一旦溫度下降，纖維狀重組結構將消失。因此，為了維持重組結構，需在降溫前先進行交聯處理。再經過冷凍乾燥後，支架明顯收縮形成薄膜。在電子顯微鏡下觀察，重組後之膠原蛋白支架，會有明顯之層狀結構，中間穿插有纖維狀結構，在支架表面，形成無孔洞的緻密層，與骨工程學的支架，需有適當大小且互通的孔洞要求，以利細胞生長，不相符合。另一種形成膠原蛋白支架的方法，是未經過重組過程，其可形成孔洞互相連接的支架。控制溫度越低，孔洞形狀越細長。-20°C的狀態下，形成多角形的孔洞。-80°C則形成較橢圓形孔洞，且孔洞大小均勻不一之程度較-20°C形成的支架為大。-196°C狀態下形成的膠原蛋白支架其孔洞則屬於細長型。-196°C狀態下形成的膠原蛋白支架，易收縮與碎裂，非常難操作，且交聯處理後，在電子顯微鏡下觀察，其孔洞消失。因此排除在以下的體內以及體外的測試操作。
體外測試方面，利用骨細胞株MC3T3-E1 subclone 4，與不同條件下形成的膠原蛋白支架一起培養。骨細胞的反應包括細胞增生數目、骨鈣素（osteocalcin）的表現與膠原蛋白支架鈣化程度。細胞增生數目是藉量測去氧核醣核酸（DNA）的含量；骨鈣素的表現則是利用RT-PCR與real time RT-PCR做檢測；鈣化程度則是應用ortho-cresolphthalein complexone method和von Kossa 染色來檢測膠原蛋白支架的鈣含量。骨細胞與不同條件下形成的膠原蛋白支架一起培養，未見有明顯的毒性反應。比較在不同溫度下（-20°C與-80°C）形成的膠原蛋白支架，包括細胞增生數目、骨鈣素的表現與膠原蛋白支架鈣化程度，皆未有統計上的明顯差異性。但是未利用戊二醛交聯處理的支架，在細胞培養過程中，支架有收縮的情況發生。利用戊二醛交聯的支架，在第七天時檢測出較多的細胞增生（p < 0.005）。然而在第七天時其骨鈣素的表現較低（p < 0.05）。在21天時支架有較少的鈣沉積（p < 0.001）。
在體內測試方面，利用大鼠頂骨雙側骨缺損模型，來檢測以不同溫度與戊二醛交聯處理之支架對於骨修補能力的影響，發現在四週、八週以及十二週的骨癒合過程，大部分的骨缺損，其骨再生量，隨癒合時間遞增而增多。在X¬-光片的檢視發現，未用戊二醛交聯處理的支架（包括-20°C與-80°C溫度處理）， 其骨缺損部位在X¬-光片上有較多放射線不透射區。利用壓克力梯尺作為參考尺，運算出的骨密度發現，未用戊二醛交聯之支架，可再生出較高的骨密度，而且有較大骨面積的形成。在組織切片檢查，戊二醛處理的支架植入之骨缺損區，在不同的時間點上，皆有明顯的骨移植物殘留，且骨移植物殘留周圍圍繞有明顯的空腔。相反的，植入未用戊二醛交聯處理支架之骨缺損則無此現象。整體來說，使用戊二醛處理的膠原蛋白支架，其新生骨較少。
總結來說，經過重組過程的膠原蛋白因結構緻密，孔洞太小，不適為骨工程用支架。利用溫度可控制孔洞大小及形狀。溫度對於支架的形成，雖可改變支架結構，卻不會影響對體外骨細胞的反應，或是體內骨缺損的修補。相反的，利用戊二醛雖然可以增進支架的強度，減少細胞培養的收縮，卻可能抑制骨細胞的分化以及鈣化作用。此外，也可能使得體內骨缺損癒合度變差。

Tissue engineering involves using scaffolds, cells and factors to regenerate organs and tissues. The application of tissue engineering strategies to bone regenerative therapies has received increasing interest in recent years. Autogenous bone has been the most preferred bone grafting material; however, its use is often limited by the availability of a source and morbidity at the donor site. Therefore, bone engineering has emerged as an alternative approach to regenerate bone.
Type I collagen is the most abundant extracellular protein in bone. Collagen sponge meets many properties of an ideal scaffold including porosity, biocompatibility, hydrophicility, biodegradability and cell recognition. During bone engineering, the osteoblastic cells grown in the scaffold undergo proliferation, differentiation and mineralization stages to form bone.
The freezing temperature and crosslinking process are two important factors to form collagen scaffolds. Crosslinking can improve mechanical strength and decrease degradation for collagen, while freezing temperature may change pore size and shape for collagen scaffold. The purpose of this study was to investigate the effects of freezing temperature and glutaraldehyde crosslinking of collagen sponges on osteoblastic responses in vitro and bony defect healing in vivo.
Type I atellocollagen was extracted from Sprague Dawley rat tails with pepsin treatment and salt precipitation. Three factors including temperature (-20°C, -80°C and -196°C), collagen reconstitution and glutaraldehyde (GA) crosslinking were used in this study to control scaffold structure and mechanical properties. After reconstitution at 37°C, white color collagen gel was formed. However, the fibrous structure of reconstitution is reversible. When lowering the temperature, the fibrous structure disappeared soon. Crosslinking is necessary before freezing for maintaining the reconstituted structure. After lyophilization of reconstituted collagen gel, membrane type scaffold was formed. Dense collagen membrane without pores on top surfaces of scaffold was observed under SEM. On the longitudinal surfaces, layered structures with intervening fibrous structure were found. The dense scaffold structure does not meet the requirement of scaffolds for bone engineering due to lack of interconnected pore structure. On the contrary, forming scaffold without reconstitutional procedure was able to form interconnected pore structure in scaffold. SEM micrographs of the -20UL (-20°C, GA uncrosslinking) and the -20L (-20°C, GA crosslinking) collagen scaffolds demonstrated a more polygonal and homogenous pore structure on both the surface facing Petri dish and the longitudinal section surface. In contrast, the -80UL (-80°C, GA uncrosslinking) and -80L (-80°C, GA crosslinking) collagen scaffolds were characterized by more pores that were elongated and inhomogeneous. NUL (-196°C, GA uncrosslinking) revealed narrow and long pore structure and was very fragile and difficult to manipulate. In addition, pore structure disappeared on NL (-196°C, GA crosslinking) under SEM observation. Due to the aforementioned characteristics, both NUL and NL were excluded in this study for further experiments.
MC3T3-E1 subclone 4 osteoblastic cells were cultured in differently prepared sponges. Osteoblastic responses examined included cell numbers, osteocalcin expression, and calcium deposition. Cell numbers were measured by DNA content. Osteocalcin expression was determined by RT-PCR and real-time RT-PCR. Calcium deposition was assayed by ortho-cresolphthalein complexone method and von Kossa stain. The osteoblastic cells grown in all collagen sponges did not show apparent signs of cytotoxicity. Collagen sponges differed in freezing temperatures resulted in similar osteoblastic responses. Glutaraldehyde-crosslinked sponges demonstrated less cell-mediated contraction and more cell numbers at day 7 (p < 0.005). However, they showed lower osteocalcin expression at day 7 (p < 0.05) and less calcium deposition at day 21 (p < 0.001).
Bilateral calvarial bony defect model of SD rats was used in this study for graft implantation. As a general rule, new bone formation increased by time. Radiographic analysis revealed apparent radiopaque area in uncrosslinked group. Bone density deduced from polynomial made by an acrylic step wedge showed higher densities in uncrosslinked group. Uncrosslinked group also showed more new bone area as well. Crosslinked group showed a lot of dense residual collagen graft materials surrounded by empty spaces or fibrous tissue under histological observation.
In summary, reconstituted collagen scaffold is not suitable for bone engineering due to dense structure and small pore size. Pore shape and size can be influenced by freezing temperature. Different freezing temperatures played a minor role in osteoblastic responses and healing of bony defect. Glutaraldehyde crosslinking process, though improved the dimensional stability of collagen sponges, might compromise the osteoblastic differentiation and mineralization in vitro. In addition, it may also lead to poorer healing of bony defect in vivo.

目錄
附圖次目錄----------------------------------------------------------------------------------------------- iii
圖次目錄--------------------------------------------------------------------------------------------------- iii
表次目錄--------------------------------------------------------------------------------------------------- iv
中文摘要--------------------------------------------------------------------------------------------------- v
英文摘要--------------------------------------------------------------------------------------------------- viii
第一章 序論--------------------------------------------------------------------------------------------- 1
1. 前言---------------------------------------------------------------------------------------- 1
1-1 文獻回顧-------------------------------------------------------------------------------- 3
1-1-1 膠原蛋白之簡介--------------------------------------------------------------------- 3
1-1-2 骨工程學的基本理論------------------------------------------------------------- 6
1-1-2-1 骨工程學材料的需求------------------------------------------------------------- 6
1-1-2-2 膠原蛋白在骨工程學上的應用---------------------------------------------- 7
1-1-3 膠原蛋白的交聯--------------------------------------------------------------------- 8
1-1-3-1 膠原蛋白體內的交聯反應----------------------------------------------------- 8
1-1-3-2 膠原蛋白體外的交聯反應----------------------------------------------------- 9
1-1-3-3 戊二醛在膠原蛋白植體所扮演的角色---------------------------------- 10
1-1-4 體外骨細胞培養的優良模型¬--MC3T3-E1 subclone 4 ----------- 10
1-2 實驗目的-------------------------------------------------------------------------------- 11
第二章 材料與方法---------------------------------------------------------------------------- 18
2-1 第一型膠原蛋白的純化--------------------------------------------------------- 18
2-2 膠原蛋白純度的鑑定------------------------------------------------------------- 19
2-3 膠原蛋白基質的製作------------------------------------------------------------- 20
2-3-1 進行重組步驟之膠原蛋白支架的製作---------------------------------- 20
2-3-2 未進行重組步驟之膠原蛋白支架的製作--溫度與交聯控制- 21
2-4 膠原蛋白支架結構的探討----------------------------------------------------- 23
2-4-1 膠原蛋白支架電子顯微鏡觀察---------------------------------------------- 23
2-4-2 膠原蛋白孔隙度分析------------------------------------------------------------- 24
2-5 MC3T3-E1 subclone 4細胞的鈣化測試--------------------------------- 24
2-6 鈣化檢測（von Kossa Stain）步驟----------------------------------------- 25
2-7 在膠原蛋白支架上培養MC3T3-E1 subclone 4細胞-------------- 26
2-8 膠原蛋白支架之體積穩定度-------------------------------------------------- 26
2-9 細胞增生的檢測--------------------------------------------------------------------- 27
2-10 細胞分化分析------------------------------------------------------------------------- 27
2-10-1 傳統RT-PCR--------------------------------------------------------------------------- 27
2-10-2 Real-time PCR------------------------------------------------------------------------ 28
2-11 鈣化分析-------------------------------------------------------------------------------- 30
2-11-1 ortho-cresolphthalein complexone方法----------------------------------- 30
2-11-2 von Kossa染色----------------------------------------------------------------------- 31
2-12 大鼠頂骨雙側骨缺損模型----------------------------------------------------- 31
2-13 放射線影像分析--------------------------------------------------------------------- 32
2-14 骨缺損的組織學觀察------------------------------------------------------------- 33
2-15 統計分析-------------------------------------------------------------------------------- 34
第三章 結果---------------------------------------------------------------------------------------- 34
3-1 膠原蛋白的純化--------------------------------------------------------------------- 34
3-2 重組處理對膠原蛋白支架的影響------------------------------------------ 34
3-3 未經重組步驟的膠原蛋白支架之結構---------------------------------- 36
3-3-1 掃描式電子顯微鏡觀察--------------------------------------------------------- 36
3-3-2 光學顯微鏡的觀察與量測----------------------------------------------------- 37
3-4 細胞的培養與形態的觀察----------------------------------------------------- 38
3-5 膠原蛋白支架體積之穩定度-------------------------------------------------- 38
3-6 不同膠原蛋白支架處理對於細胞增生的影響----------------------- 40
3-7 不同膠原蛋白支架處理對於細胞分化的影響----------------------- 40
3-8 不同膠原蛋白支架處理對於鈣沈積的影響--------------------------- 41
3-9 手術過程與觀察--------------------------------------------------------------------- 42
3-10 放射線攝影之觀察----------------------------------------------------------------- 43
3-11 放射線分析---------------------------------------------------------------------------- 44
3-11-1 骨密度------------------------------------------------------------------------------------ 44
3-11-2 新骨面積-------------------------------------------------------------------------------- 45
3-12 手術區域的組織學觀察--------------------------------------------------------- 46
第四章 討論---------------------------------------------------------------------------------------- 48
第五章 結論---------------------------------------------------------------------------------------- 57
參考資料 ------------------------------------------------------------------------------------------------ 94



附圖次目錄
圖1 膠原蛋白三股螺旋結構--------------------------------------------------------- 15
圖2 戊二醛的化學式--------------------------------------------------------------------- 16
圖3 膠原蛋白以戊二醛化學交聯之示意圖---------------------------------- 16
圖4 MC3T3-E1 subclone 4細胞往骨細胞分化時間表------------------ 17
圖5 由大鼠萃取第一型膠原蛋白步驟------------------------------------------ 22
圖6 RNA的萃取步驟流程------------------------------------------------------------ 29



圖次目錄
圖7 大鼠頂骨對稱的骨缺損圖----------------------------------------------------- 64
圖8 電泳膠片圖---------------------------------------------------------------------------- 64
圖9 膠原蛋白不同的型態------------------------------------------------------------- 65
圖10 膠原蛋白支架電顯圖：重組後，在不同的溫度下作交聯---- 66
圖11 膠原蛋白支架電顯圖：不同重組時間------------------------------------ 67
圖12 膠原蛋白支架電顯圖：不同GA濃度交聯，2 %膠原蛋白------- 68
圖13 膠原蛋白支架電顯圖：不同GA濃度交聯，1 %膠原蛋白------- 69
圖14 膠原蛋白支架電顯圖：不同交聯時間，2 %膠原蛋白---------- 70
圖15 膠原蛋白支架電顯圖：不同交聯時間，1 %膠原蛋白---------- 71
圖15-1 膠原蛋白支架電顯圖（內部分層）--------------------------------------- 72
圖16 膠原蛋白支架電顯圖：-20UL，-20L，-80UL，-80L -------------- 73
圖17 膠原蛋白支架電顯圖：NUL，NL ---------------------------------------- 73
圖18 膠原蛋白支架光學顯微鏡圖-------------------------------------------------- 74
圖19 MC4細胞光學顯微鏡圖--------------------------------------------------------- 75
圖20 MC4細胞電顯圖-------------------------------------------------------------------- 76
圖21 膠原蛋白支架直徑表示圖----------------------------------------------------- 77
圖22 細胞數表示圖------------------------------------------------------------------------- 78
圖23 骨鈣素表示圖：RT-PCR---------------------------------------------------------- 79
圖24 骨鈣素表示圖：Real-time RT-PCR------------------------------------------ 80
圖25 鈣含量表示圖------------------------------------------------------------------------- 81
圖26 von Kossa染色組織切片圖---------------------------------------------------- 82
圖27 手術區頂骨底下鄰接的大腦組織------------------------------------------ 83
圖28 頂骨X-光片圖------------------------------------------------------------------------ 84
圖29 二次多項式圖------------------------------------------------------------------------- 85
圖30 骨密度表示圖------------------------------------------------------------------------- 86
圖31 新生骨面積表示圖----------------------------------------------------------------- 87
圖32 術後4週的組織切片圖：-20UL，-20L---------------------------------- 88
圖33 術後4週的組織切片圖：-80UL，-80L---------------------------------- 89
圖34 術後8週的組織切片圖：-20UL，-20L---------------------------------- 90
圖35 術後8週的組織切片圖：-80UL，-80L---------------------------------- 91
圖36 術後12週的組織切片圖：-20UL，-20L-------------------------------- 92
圖37 術後12週的組織切片圖：-80UL，-80L-------------------------------- 93



表次目錄
表1 主要的膠原蛋白類型與其功能---------------------------------------------- 12
表2 骨工程支架設計的考慮要點-------------------------------------------------- 13
表3 不同交聯方法的比較------------------------------------------------------------- 14
表4 樣品緩衝液（Sample buffer）的配製------------------------------------ 58
表5 電泳膠片的配製--------------------------------------------------------------------- 59
表6 染色液與去染色液的配製----------------------------------------------------- 59
表7 重組膠原蛋白支架之控制變數---------------------------------------------- 60
表8 膠原蛋白支架之分類------------------------------------------------------------- 60
表9 DNase I 的處理程序-------------------------------------------------------------- 61
表10 RT步驟----------------------------------------------------------------------------------- 61
表11 聚合酶鍊反應之條件------------------------------------------------------------- 62
表12 聚合酶鍊反應之primer---------------------------------------------------------- 62
表13 骨密度的統計表--------------------------------------------------------------------- 63
表14 骨面積的統計表--------------------------------------------------------------------- 63

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