透明質酸及含氫氧基磷灰石之膠原蛋白複合基質對於脂肪基質細胞增生及骨分化潛能之效應

Abstract

適當的細胞來源和擴增技術，以及良好的支架對於組織工程的應用而言極為重要。而本研究即以骨組織工程為目標，針對細胞的來源和培養技術，以及促骨組織工程支架之製備進行深入地研究和探討。首先，進行脂肪基質細胞之分離純化，並探討其在體外擴增之可行性；繼而開發一種能提供細胞生長、遷移進而能引導及誘導骨質新生之組織工程支架，以其能使用於骨骼重建中。 首先，本研究使用透明質酸進行小鼠脂肪基質細胞之培養，並探討不同之含透明質酸培養環境對於細胞生理反應之影響。由實驗結果可知，添加透明質酸於培養液中可促進基質細胞增生、延緩因連續繼代所造成之老化現象、並增進其骨分化之潛能。另一方面，經透明質酸塗覆之表面則可使基質細胞進入細胞週期之靜止期，後續將此靜止狀態之細胞轉移至一般培養環境下培養後，基質細胞之增生能力及分化潛能立即恢復，且可延長其在體外之增生期限。因此，透明質酸之塗覆表面不但可避免基質細胞之老化，而且亦能維持其增生活性及分化潛能。 其次，由於羥基磷灰石為骨質無機鹽類中之主要成份，可促進骨細胞之增生及分化；膠原蛋白為胞外基質中重要的結構性蛋白，具有良好之生物相容性，使其成為常用之細胞培養基材；而透明質酸則為胞外基質中主要的葡萄胺聚醣，其具有調控細胞增生、遷移、分化的諸多功能，且在胚胎發育、組織成型以及傷口癒合等層面中，扮演及為重要的角色。因此，結合羥基磷灰石、透明質酸及膠原蛋白之多孔性基質應可作為體外骨細胞培養以及體內骨組織修復之理想模板。 因此，本研究藉由濕式研磨法將羥基磷灰石研磨成奈米微粒，將其與膠原蛋白溶液混合，並在該溶液中添加能調控許多細胞生理反應之高分子量透明質酸，進而以相分離法製備出含奈米羥基磷灰石及透明質酸之膠原蛋白多孔性基質。經實驗證實，此種多孔性複合基質極適合細胞在體外增生，且其可在不含誘導培養液之情況下使具多向分化能力之小鼠脂肪基質細胞在體外展現出骨分化潛能。

Suitable cell sources with good expansion technique, as well as excellent scaffolds are important for tissue engineering applicability. In this study, we aimed at the application of bone tissue engineering that the feasibility of in vitro expansion of adipose-derived stromal cells was investigated, and a tissue-engineered scaffold with osteoinductive characteristics was also developed. First of all, hyaluronan was applied in this study to investigate its effect on the behaviors of mADSCs. It was shown in our results that as a medium supplement, hyaluronan could promote the proliferation and differentiation of mADSCs, and delay the build-up of senescence programs as well. On the other hand, upon culturing on hyaluronan-coated surface, mADSCs seemed to be quiescent without losing its proliferative and differentiation capacity as shown from the results that when a transfer of culture from quiescent state to regular culture environment was performed, the proliferative and differentiation potentials were restored, indicating that hyaluronan-coated surface could prevent the senescence and maintain the proliferation and differentiation of mADSCs. In addition, being the predominant component of the inorganic substances in bone matrix, hydroxyapatite was proved to promote the proliferation and differentiation of bone cells. Collagen, the important structural protein of extracellular matrix, has been broadly utilized to prepare cell culture scaffolds due to its excellent biocompatibility. The porous matrix composed of both hydroxyapatite and collagen can therefore be used in bone cell culture in vitro as well as bone tissue regeneration in vivo. Thus, we were instigated to fabricate the hydroxyapatite nanoparticles by wet-milling and they were then combined with collagen and hyaluronan which was capable of regulating various cell physiological behaviors to form a porous scaffold via phase separation technique. Our results demonstrated that the porous hydroxyapatite/hyaluronan/collagen composite matrix was suitable for cell proliferation in vitro, and it could also possess the osteoinductive effect that it was shown to promote the differentiation of murine adipose-derived stromal cells (mADSCs) towards osteogenic lineage without the administration of induction medium.