Olvera Dinorath, Sathy Binulal N, Carroll Simon F, Kelly Daniel J
Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland.
Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland; Tissue Engineering Research Group, Department of Anatomy, Royal College of Surgeons in Ireland, Dublin 2, Ireland; Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin, Dublin 2, Ireland.
Acta Biomater. 2017 Dec;64:148-160. doi: 10.1016/j.actbio.2017.10.010. Epub 2017 Oct 7.
The ideal tissue engineering (TE) strategy for ligament regeneration should recapitulate the bone - calcified cartilage - fibrocartilage - soft tissue interface. Aligned electrospun-fibers have been shown to guide the deposition of a highly organized extracellular matrix (ECM) necessary for ligament TE. However, recapitulating the different tissues observed in the bone-ligament interface using such constructs remains a challenge. This study aimed to explore how fiber alignment and growth factor stimulation interact to regulate the chondrogenic and ligamentous differentiation of mesenchymal stem cells (MSCs). To this end aligned and randomly-aligned electrospun microfibrillar scaffolds were seeded with bone marrow derived MSCs and stimulated with transforming growth factor β3 (TGFβ3) or connective tissue growth factor (CTGF), either individually or sequentially. Without growth factor stimulation, MSCs on aligned-microfibers showed higher levels of tenomodulin (TNMD) and aggrecan gene expression compared to MSCs on randomly-oriented fibers. MSCs on aligned-microfibers stimulated with TGFβ3 formed cellular aggregates and underwent robust chondrogenesis, evidenced by increased type II collagen expression and sulphated glycosaminoglycans (sGAG) synthesis compared to MSCs on randomly-oriented scaffolds. Bone morphogenetic protein 2 (BMP2) and type I collagen gene expression were higher on randomly-oriented scaffolds stimulated with TGFβ3, suggesting this substrate was more supportive of an endochondral phenotype. In the presence of CTGF, MSCs underwent ligamentous differentiation, with increased TNMD expression on aligned compared to randomly aligned scaffolds. Upon sequential growth factor stimulation, MSCs expressed types I and II collagen and deposited higher overall levels of collagen compared to scaffolds stimulated with either growth factor in isolation. These findings demonstrate that modulating the alignment of microfibrillar scaffolds can be used to promote either an endochondral, chondrogenic, fibrochondrogenic or ligamentous MSC phenotype upon presentation of appropriate biochemical cues.
Polymeric electrospun fibers can be tuned to match the fibrillar size and anisotropy of collagen fibers in ligaments, and can be mechanically competent. Therefore, their use is attractive when attempting to tissue engineer the bone-ligament interface. A central challenge in this field is recapitulating the cellular phenotypes observed across the bone-ligament interface. Here we demonstrated that it is possible to direct MSCs seeded onto aligned electrospun fibres towards either a ligamentogenic, chondrogenic or fibrochondrogenic phenotype upon presentation of appropriate biochemical cues. This opens the possibility of using aligned microfibrillar scaffolds that are spatially functionalized with specific growth factors to direct MSC differentiation for engineering the bone-ligament interface.
用于韧带再生的理想组织工程（TE）策略应重现骨 - 钙化软骨 - 纤维软骨 - 软组织界面。已证明排列整齐的电纺纤维可引导韧带组织工程所需的高度有序细胞外基质（ECM）的沉积。然而，使用此类构建体重现骨 - 韧带界面中观察到的不同组织仍然是一项挑战。本研究旨在探讨纤维排列和生长因子刺激如何相互作用以调节间充质干细胞（MSC）的软骨生成和韧带分化。为此，将骨髓来源的MSC接种到排列整齐和随机排列的电纺微纤维支架上，并分别或依次用转化生长因子β3（TGFβ3）或结缔组织生长因子（CTGF）刺激。在没有生长因子刺激的情况下，与随机取向纤维上的MSC相比，排列整齐的微纤维上的MSC显示出更高水平的腱调蛋白（TNMD）和聚集蛋白聚糖基因表达。用TGFβ3刺激的排列整齐的微纤维上的MSC形成细胞聚集体并经历强大的软骨生成，与随机取向支架上的MSC相比，II型胶原蛋白表达增加和硫酸化糖胺聚糖（sGAG）合成证明了这一点。在用TGFβ3刺激的随机取向支架上，骨形态发生蛋白2（BMP2）和I型胶原蛋白基因表达更高，表明该底物更支持软骨内表型。在CTGF存在下，MSC经历韧带分化，与随机排列的支架相比，排列整齐的支架上TNMD表达增加。在顺序生长因子刺激下，与单独用任何一种生长因子刺激的支架相比，MSC表达I型和II型胶原蛋白并沉积更高的总体胶原蛋白水平。这些发现表明，在呈现适当的生化线索时，调节微纤维支架的排列可用于促进软骨内、软骨生成、纤维软骨生成或韧带性MSC表型。
聚合物电纺纤维可以调整以匹配韧带中胶原纤维的纤维尺寸和各向异性，并且在机械上具有活性。因此，在尝试对骨 - 韧带界面进行组织工程时，它们的使用具有吸引力。该领域的一个核心挑战是重现骨 - 韧带界面中观察到的细胞表型。在这里，我们证明了在呈现适当的生化线索时，可以将接种到排列整齐的电纺纤维上的MSC引导至韧带生成、软骨生成或纤维软骨生成表型。这开启了使用用特定生长因子进行空间功能化的排列整齐的微纤维支架来指导MSC分化以工程化骨 - 韧带界面的可能性。
