以高靜水壓刺激部分經酵素分解的軟骨組織塊生長之體外研究

Abstract

在關節軟骨中，軟骨細胞係被一層細胞外間質所包圍，該細胞外間質包括有水，可溶性成分以及不可溶性之聚合物。靜水壓力是已知能影響關節軟骨之發展，成熟與衰退的物理性刺激之一。先前的動物或組織工程研究都顯示隨時間變化的動態靜水壓力負荷可以促進細胞外間質的合成與衰退，其主要參數包括壓力值，頻率，培養期間與三維環境培養。在本研究中，一種新型的靜水壓力生物反應器被設計來培養經部分酵素分解之軟骨組織塊。 在酵素分解的過程中，傳統方法是使用手術刀片將組織試片在培養皿上切碎，緊接著再將組織碎塊置於酵素中分解，並實際應用於細胞培養。然而此一過程是相當容易受到污染且難以收集的。在本研究中成功地發明了一個器械能將切碎，過篩及收集在一個密閉的系統內操作完成。此外，由於切碎機能切碎組織試片卻不損傷細胞本身，故可實際應用於組織工程的細胞培養中。 在經過靜水壓力培養兩週後，在完全未經酵素分解與部分經酵素分解的軟骨組織塊其細胞能成長並穩定地分泌細胞外間質，其中包括了GAGs與第二型膠原蛋白。總結來說，經過新型靜水壓力生物反應器培養的組織型態的確更為接近天然軟骨。

In Articular cartilage, chondrocytes are embedded in an extracellular matrix composed of water, soluble constituents, and insoluble polymers. The physical stimulation of hydrostatic pressure is known to influence the development, maturation, and degeneration of cartilage. Previous animal and tissue explant studies have shown that time-varying dynamic tissue loading can increase the synthesis and deposition of matrix molecules in an amplitude, frequency, duration ands three-dimensional culture. In this study, a novel hydrostatic pressure bioreactor system was designed for culture partially digested cartilage tissue fragments. In conventional culture preparation, a tissue piece is cut in a culture dish by scalpel blade, soaked in protease enzyme to separate cells from cytoplasm, and applied to a culture experiment. When cut by scalpel blade, however, the tissue piece is easily polluted and difficult to collect. The present invention successively cuts, filters, and collects finally the homogenized tissue pieces in a sealed device. As well, because the present invention homogenizes the tissue piece by cutting, histiocytes are undamaged and can be applied in live histiocyte culture. Our result demonstrate that this hydrostatic bioreactor system enables chondrocytes under hydrostatic pressure after 2 weeks to grow and secrete extracelluar matrix comprised GAGs and type II collagen steadily. In summary , the morphologies of many cells stimulated by hydrostatic pressure were similar to the chondrocytes in normal cartilage.