富含血小板纖維蛋白在軟骨組織工程中作為生物降解性細胞支架的製備與應用

軟骨組織內缺少血管與神經，自我修復能力有限，因此對於軟骨損壞的治療上是一大挑戰。骨關節炎 (Osteoarthritis，OA)，是一種局部性關節軟骨退化病變，主因為關節軟骨組織新生合成速度趕不上分解破壞速度所導致，普遍好發於過度負重及老化。本篇論文研究目的在利用軟骨組織工程原理，以 Platelet-rich fibrin (PRF) 合併 fibrin 方式開發新型細胞生長支架 (scaffold) ，於體外培養出健康軟骨細胞，再經由注射方式修復受損之關節軟骨，探討此 fibrin + PRF 複合生長支架對於軟骨細胞的增生以及分化能力有否有所助益。富含血小板纖維蛋白 (PRF) 是近年來新發現由血液中萃取的血小板濃縮物，本身富含生長因子和細胞刺激素，尤其是 PDGF-BB、IGF-1、TGF-??1、BMP-2，這些蛋白對於軟骨細胞的增生與分化有所助益。實驗中製作了兩種 fibrin scaffold ，一種是以纖維蛋白原 (fibrinogen) 和凝血酶 (thrombin) 合成 fibrin gel，另外以 PRF exudates 作為 fibrin gel scaffold 的添加劑，生成 fibrin + PRF 複合生長支架。此外 fibrin scaffold 作成兩種型態，膠體形式與以冷凍乾燥方式做成海綿狀物質。實驗裡應用人類軟骨瘤細胞株 (human chondrosacrcoma cell, SW-1353) 和初代人類軟骨細胞 (primary human chondrocyte) 培養模式，將細胞分別培養在 fibrin gel、添加 PRF 溶液之 fibrin 複合膠體以及 agarose gel 的二維 (2D) 與三維 (3D) 三種不同生長支架中，評估軟骨細胞的增生與分化情形。實驗中發現從血液中分離所得 PRF 降解後分泌液與軟骨細胞生長相關生長因子濃度平均高於其他血液裡萃取之衍生物 (serum, plasma, fibrin)。我們由觀察外觀和使用電子顯微鏡 (SEM) 比較兩種型態 fibrin sponge scaffold 內部結構。結果顯示 gel 狀 fibrin scaffold 較 sponge 狀結構來的堅固且細胞生長效果較佳。然而軟骨細胞在 agarose gel scaffold 中因不貼附所以生長效果最差。細胞培養在三種不同 scaffold 中經過4、8、12天三個時間點作觀察，發現軟骨細胞在添加 PRF exudates 後的 fibrin scaffold 上 type II collagen 與 aggrecan 的 mRNA 表現量較純粹 fibrin scaffold 有明顯上升的趨勢，證實軟骨細胞利用此複合 3D 生長支架 re-differentiation 的效果更好。組織切片方面，經由 Alcian blue 與 PAS 染色結果得知，培養於 3D fibrin + PRF scaffold 中軟骨細胞產生之蛋白多醣 (proteoglycan) 與糖胺聚醣 (glycosaminoglycan，GAG) 量優於在 fibrin scaffold 中。綜合結果顯示出 PRF 因本身富含生長因子與細胞刺激素，使添加 PRF 後的 fibrin gel，更能促進軟骨細胞的增生及分化。未來在組織工程中，將 PRF 運用於軟骨修復上，搭配 fibrin gel 作為有效的立體生長支架使軟骨細胞能有效增生且維持細胞於分化狀態，進而達到軟骨再生效果，用於治療 OA 是具有潛力的。

關鍵字

骨關節炎；軟骨細胞；細胞支架；纖維蛋白；富含血小板纖維蛋白；瓊膠；糖胺聚醣；聚蛋白多醣；膠原蛋白

並列摘要

Because of articular cartilage has a limited ability for self- repair. Thus, treatment of cartilage lesions is a challenge. Osteoarthritis (OA), is known as degenerative joint disease and a type of arthritis that is caused by the breakdown and eventual loss of the cartilage of one or more joints. This thesis investigates to incorporate PRF that involves developmental signals into fibrin for use in cell biological studies and as a regeneration matrix employing tissue-engineering (TE). The goal is to explore the fibrin gel that adds PRF scaffold for in vitro culture and in vivo of chondrocytes proliferation and differentiation. Platelet-rich fibrin (PRF) is a new generation of platelet concentration, it’s abundant in platelet cytokines, like platelet-derived growth factors (PDGF-BB), insulin-like growth factor-1 (IGF-1), transforming growth factor (TGF-??1) and bone morphogenetic proteins (BMP-2). They have the ability of chondrocytes proliferation and differentiation. In the thesis, fibrin scaffolds have two types. One is produced from bovine fibrinogen and thrombin that mix to fabricate the fibrin gel, and the other is add PRF from human blood by centrifugation without anticoagulant into fibrin scaffold. And the fibrins are made two forms, the fibrin gel and it is to make a fibrin sponge by freeze-drying. We set up the primary human chondrocytes and SW-1353 . The primary chondrocytes and SW-1353 are embedded and developed in these scaffolds. Additionally, the agarose scaffold is to be the control. Three types of three-dimension (3D) and two-dimension (2D) culture are fibrin, fibrin with PRF and agarose scaffold are evaluated，which is the adaptive one to enable the chondrocytes proliferation and differentiation . We compare SW-1353 and primary human chondrocytes on 2D and in 3D of three kinds of scaffold. In order to carry out a comparative study, we undertake the inner microstructure of fibrin, PRF sponge that has an interconnected pore structure is observed by scanning electron microscope (SEM). And we observe cell morphology by using microscopy. To quantify the concentration of growth factors in the PRF exduates, PDGF-BB, IGF-1, TGF-??1, and BMP-2 are stimulators that correlated with proliferation and differentiation by ELISA. MTT assay and RT-PCR are used for estimate cell survivability and the mRNA expression of type II collagen and aggrecan. To detect the glycosaminoglycan (GAG) from chondrocytes are in different scaffold by PAS and Alician blue staining. The results showed that the chondrocytes on 2D and in 3D fibrin + PRF scaffold structure could provided the more available proliferation and differentiation than cells just on 2D and in 3D fibrin or agarose. It is concluded that the fibrin with PRF gel including abundant cytokines and growth factors is a promising three-dimension scaffold of cells for cartilage tissue engineering.

並列關鍵字

osteoarthritis ； chondrocyte ； scaffold ； fibrin ； platelet-rich fibrin ； agarose ； glycosaminoglycan ； aggrecan ； collagen

參考文獻

1. Dr R. L. Mauck Ph.D., X. Yuan and Dr R. S. Tuan Ph.D., Branch Chief. Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthr. Cartilage 2006 ;14: 179-189

2. Martin Fussenegger, MD, Johann Meinhart, PhD,Walter Höbling, MD, Werner Kullich, PhD, Siegfried Funk, MD, and Gu¨nther Bernatzky, PhD. Stabilized autologous fibrin-chondrocyte constructs for cartilage repair in vivo. Ann. Plast. Surg 2003;51: 493–498.

3. Adam Curtis and Mathis Riehle. Tissue engineering: the biophysical background. Phys. Med. Biol. 2001;46: 47–65.

4. Castern Perka, MD., Ulrike Arnold, MD., Ron-Sascha Spitzer, MD, and Klaus Lindenhayn, PhD. The use of fibrin beads for tissue engineering and subsequential transplantation. Tissue. Eng Volume 7, Number 3, 2001.

5. Elvira Malicev, Damjan Radosavljevic, Nevenka Kregar Velikonja. Fibrin gel improved the spatial uniformity and phenotype of human chondrocytes seeded on collagen scaffolds. Biotechnol. Bioeng 2007;96: 364–370.

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富含血小板纖維蛋白在軟骨組織工程中作為生物降解性細胞支架的製備與應用

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