1 Department of Orthopaedic Surgery, Duke University Medical Center , Durham, North Carolina.
Tissue Eng Part A. 2013 Dec;19(23-24):2594-604. doi: 10.1089/ten.TEA.2013.0165. Epub 2013 Aug 29.
Full-thickness rotator cuff tears are one of the most common causes of shoulder pain in people over the age of 65. High retear rates and poor functional outcomes are common after surgical repair, and currently available extracellular matrix scaffold patches have limited abilities to enhance new tendon formation. In this regard, tissue-engineered scaffolds may provide a means to improve repair of rotator cuff tears. Electrospinning provides a versatile method for creating nanofibrous scaffolds with controlled architectures, but several challenges remain in its application to tissue engineering, such as cell infiltration through the full thickness of the scaffold as well as control of cell growth and differentiation. Previous studies have shown that ligament-derived extracellular matrix may enhance differentiation toward a tendon or ligament phenotype by human adipose stem cells (hASCs). In this study, we investigated the use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaffolds compared to fibronectin (FN) or phosphate-buffered saline (PBS) coating for use in rotator cuff tendon tissue engineering. Multilayered poly(ɛ-caprolactone) scaffolds were prepared by sequentially collecting electrospun layers onto the surface of a grounded saline solution into a single scaffold. Scaffolds were then coated with TDM, FN, or PBS and seeded with hASCs. Scaffolds were maintained without exogenous growth factors for 28 days in culture and evaluated for protein content (by immunofluorescence and biochemical assay), markers of tendon differentiation, and tensile mechanical properties. The collagen content was greatest by day 28 in TDM-scaffolds. Gene expression of type I collagen, decorin, and tenascin C increased over time, with no effect of scaffold coating. Sulfated glycosaminoglycan and dsDNA contents increased over time in culture, but there was no effect of scaffold coating. The Young's modulus did not change over time, but yield strain increased with time in culture. Histology demonstrated cell infiltration through the full thickness of all scaffolds and immunofluorescence demonstrated greater expression of type I, but not type III collagen through the full thickness of the scaffold in TDM-scaffolds compared to other treatment groups. Together, these data suggest that nonaligned multilayered electrospun scaffolds permit tenogenic differentiation by hASCs and that TDM may promote some aspects of this differentiation.
全层肩袖撕裂是 65 岁以上人群肩部疼痛的最常见原因之一。手术后再撕裂率和功能结果不佳较为常见，目前可用的细胞外基质支架贴片增强新肌腱形成的能力有限。在这方面，组织工程支架可能提供一种改善肩袖撕裂修复的方法。静电纺丝提供了一种具有可控结构的纳米纤维支架的多功能方法，但在其应用于组织工程方面仍存在一些挑战，例如细胞渗透整个支架的厚度以及控制细胞生长和分化。以前的研究表明，韧带衍生的细胞外基质可以通过人脂肪干细胞（hASC）增强向肌腱或韧带表型的分化。在这项研究中，我们研究了与纤维连接蛋白（FN）或磷酸盐缓冲盐水（PBS）涂层相比，肌腱衍生细胞外基质（TDM）涂层的静电纺丝多层支架在肩袖肌腱组织工程中的应用。多层聚（己内酯）支架通过将静电纺丝层顺序收集到生理盐水表面上的单个支架中来制备。然后将支架用 TDM、FN 或 PBS 涂层并接种 hASC。在没有外源性生长因子的情况下，将支架在培养中维持 28 天，并评估蛋白质含量（通过免疫荧光和生化测定）、肌腱分化标志物和拉伸力学性能。在 TDM 支架中，第 28 天胶原含量最高。I 型胶原、饰胶蛋白聚糖和腱糖蛋白 C 的基因表达随时间增加，支架涂层没有影响。硫酸化糖胺聚糖和 dsDNA 含量随时间增加，但支架涂层没有影响。杨氏模量随时间没有变化，但屈服应变随培养时间增加。组织学显示所有支架的全厚度细胞渗透，免疫荧光显示 TDM 支架中 I 型但不是 III 型胶原的全厚度表达大于其他治疗组。综上所述，这些数据表明，非定向多层静电纺丝支架允许 hASC 产生腱细胞分化，TDM 可能促进这种分化的某些方面。
