利用重組桿狀病毒改質脂肪間葉幹細胞應用於軟骨組織工程

Applications of Baculovirus-Mediated Genetic Modification of Adipose-Derived Mesenchymal Stem Cells in Cartilage Tissue Engineering

盧佳昕(Chia-Hsin Lu)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/博士(2012年)

摘要

脂肪間葉幹細胞(adipose-derived mesenchymal stem cells, ASCs)為多潛能型幹細胞，具有分化成軟骨細胞之潛力且容易自脂肪組織分離，現已廣泛應用於組織工程。然而，脂肪間葉幹細胞的軟骨分化效率與骨髓間葉幹細胞相比卻明顯不足。我們假設若能將刺激ASCs軟骨化的生長因子濃度增加，並培養於低氧氣濃度以及動態培養環境下，將有利於ASCs分化為軟骨細胞(chondrocytes)，並且在體外培養時形成工程軟骨(engineered cartilage)。 因此，本研究之初期目的便是利用重組桿狀病毒(Bac-CT3W)表現轉化生長因子(transforming growth factor-β3, TGF-β3)來改質ASCs，並培養於兩相旋轉軸生物反應器(rotating shaft bioreactor, RSB)中，期望結合生物、環境與物理刺激能夠促進ASCs分化成軟骨細胞，並形成工程軟骨。我們證實經由Bac-CT3W轉導過的ASCs有劑量依賴性且能夠表現高濃度的TGF-β3生長因子。基因改質後的ASCs接種於聚乳酸-甘醇酸(Poly(Lactide-co-Glycolide), PLGA)細胞支架並在RSB反應器中培養兩週後，細胞形態類似軟骨細胞，並可以在細胞/支架結構物的表面及周圍產生大量的軟骨特異性細胞外間質，如葡萄糖胺聚醣(glycosaminoglycans, GAGs)與第二型膠原蛋白(collagen type II, COL II)。當培養週數超過兩週，結構物的細胞外基質會減少，可能是因為細胞外基質的分子分解所導致。總結來說，結合重組桿狀病毒基因改質的ASCs與RSB的動態培養能夠提高ASCs的軟骨分化效果並形成類軟骨組織，但培養時間卻是形成優化的工程軟骨之重要關鍵因素。
由於ASCs於體外分化緩慢，因此我們假設若能對ASCs長期提供生長因子刺激以利於體外培養形成軟骨組織，將有利於培養出更接近天然軟骨組織之工程軟骨並加速其體外培養的時間和加強工程軟骨的體外培養效果。因此，我們利用能夠長效表現TGF-β3和第六骨形態發生蛋白質(bone morphological protein-6, BMP-6)的混成桿狀病毒來改質ASCs，希望利用具有抑制軟骨細胞肥大化(hypertrophy)能力的TGF-β3和能增強ASCs細胞表面之轉化生長因子受體-I (TGF-β receptor I)表現的BMP-6之協同作用，增強刺激ASCs往軟骨化路徑分化的效果。我們將基因改質後的ASCs植入已由明膠(gelatin)、透明質酸(hyaluronic acid, HA)和硫酸軟骨素(chondroitin sulfate, CS)共聚合接枝在PLGA支架上而成的PLGA-GCH細胞支架中，並培養於接近天然軟骨環境之低氧氣濃度條件。實驗結果證實，經由混成長效型重組桿狀病毒表現系統改質之ASCs在3D立體培養環境下能維持混成生長因子群之高濃度，刺激ASCs往軟骨細胞路徑分化並表現軟骨標誌基因(SOX 9、Aggrecan和COL2A1)，抑制纖維軟骨標誌基因(COL1A2)與細胞肥大化標誌基因(COL10A1)之表現，且工程軟骨產生大量的軟骨特異性細胞外間質如GAGs與COL II。體外培養兩週之工程軟骨在植入紐西蘭白兔的膝關節負重缺陷處後，以磁振造影(magnetic resonance imaging, MRI)能清楚的觀察到以混成長效型表現系統培養之工程軟骨能在術後12週即達到良好的軟骨組織修復效果。本研究是首次結合新穎的長效型表現生長因子之重組桿狀病毒改質的ASCs與PLGA-GCH細胞支架，並於體外環境下培養出工程軟骨，應用於兔膝關節負重區之軟骨組織修復，本實驗結果證實混成長效型表現系統於軟骨組織工程領域具有高度的研究潛力且具有未來臨床應用價值。

關鍵字

桿狀病毒 ； 轉化生長因子β3 ； 第六骨形態發生蛋白質 ； 工程軟骨 ； 組織工程

並列摘要

Adipose-derived mesenchymal stem cells (ASCs) are easy to isolate from adipose tissues in abundance and have been widely employed in tissue engineering. However, ASCs appear to be inferior to bone marrow mesenchymal stem cells in terms of chondrogenic potential. We hypothesize that proper supply of transforming growth factor, low oxygen concentration and a dynamic environment could facilitate ASCs to differentiate into chondrocytes and to promote the engineered cartilage formation. Therefore, in this study we first developed a baculoviral vector that expressed TGF-β3 (Bac-CT3W) for the genetic modification of ASCs, which were then cultured in the two-phase rotating shaft bioreactor (RSB), hoping that the biological, environmental and physical stimuli altogether can facilitate the ASCs differentiation into chondrocytes and growth into engineered cartilages in vitro. We demonstrated that ASCs transduced with Bac-CT3W expressed high levels of TGF-β3 in a dose-dependent manner. After seeding the cells into 3D, porous scaffolds and culture in the RSB for 2 weeks, the cell/scaffold constructs grew into cartilage-like tissues with hyaline appearance and the cells resembled the authentic chondrocytes. The constructs were abundant in cartilage-specific extracellular matrix (ECM) including collagen II and glycosaminoglycan as demonstrated by histological staining and biochemical assays. Intriguingly, when the construct culture time exceeded 2 weeks, the abundance of ECM decreased, probably due to the degradation of the ECM molecules. These data demonstrated that combining the TGF-β3-expressing baculovirus and RSB for the genetic modification and dynamic culture of ASC enhances the chondrogenic differentiation of ASCs and cartilage formation, but the culture time appears to be critical to the cartilage quality.
The chondrogenic differentiation of ASCs is time-consuming and requires sustained growth factor expression. We further developed hybrid baculoviruses that can persistently express TGF-β3 and bone morphological protein-6 (BMP-6) to genetically modify ASCs, hoping that TGF-β3 can suppress the hypertrophy of chondrocytes while BMP-6 can further enhance the chondrogenesis of ASCs. Furthermore, we developed new scaffolds that comprised PLGA, gelatin, chondroitin sulfate and hyaluronic acid (PLGA-GCH), hoping that these components futher synergistically promote ASCs chondrogenesis. The genetically engineered ASCs were seeded to the porous PLGA-GCH scaffolds and cultivated under low oxygen concentration condition that mimic the native cartilage environment. We demonstrated that transduction of ASCs with the hybrid baculovirus enabled sustained expression of TGF-β3 and BMP-6 at high levels in the 3D environment, stimulated the chondrogenesis of ASCs and formation of cartilage-like tissues. Transplantation of the engineered constructs into the osteochondral defects at the weight-bearing area of medial femoral condyle of rabbit knees accelerated and ameliorated the cartilage repair in 12 weeks, as evidenced by MRI. This study, for the first time, combines PLGA-GCH scaffolds and engineered ASCs persistently expressing grwoth factors for the formation of engineered cartilages and in vivo repair of osteochondral defects.

並列關鍵字

Baculovirus ； TGF-b3 ； BMP-6 ； Engineered Cartilage ； Tissue Engineering

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