Effects of Cell Seeding and Cyclic Stretch on the Fiber Remodeling in an Extracellular Matrix–Derived Bioscaffold

Abstract

The porcine small intestine submucosa, an extracellular matrix–derived bioscaffold (ECM-SIS), has been successfully used to enhance the healing of ligaments and tendons. Since the collagen fibers of ECM-SIS have an orientation of ± 30°, its application in improving the healing of the parallel-fibered ligament and tendon may not be optimal. Therefore, the objective was to improve the collagen fiber alignment of ECM-SIS in vitro with fibroblast seeding and cyclic stretch. The hypothesis was that with the synergistic effects of cell seeding and mechanical stimuli, the collagen fibers in the ECM-SIS can be remodeled and aligned, making it an improved bioscaffold with enhanced conductive properties. Three experimental groups were established: group I (n = 14), ECM-SIS was seeded with fibroblasts and cyclically stretched; group II (n = 13), ECM-SIS was seeded with fibroblasts but not cyclically stretched; and group III (n = 8), ECM-SIS was not seeded with fibroblasts but cyclically stretched. After 5 days’ experiments, the scaffolds from all the three groups (n = 9 for group I; n = 8 for groups II and III) were processed for quantification of the collagen fiber orientation with a small-angle light scattering (SALS) system. For groups I and II, in which the scaffolds were seeded with fibroblasts, the cell morphology and orientation and newly produced collagen fibrils were examined with confocal fluorescent microscopy (n =3/group) and transmission electronic microscopy (n =2/group). The results revealed that the collagen fiber orientation in group I was more aligned closer to the stretching direction when compared to the other two groups. The mean angle decreased from 25.3° to 7.1° ( p < 0.05), and the associated angular dispersion was also reduced (37.4° vs. 18.5°, p < 0.05). In contrast, groups II and III demonstrated minimal changes. The cells in group I were more aligned in the stretching direction than those in group II. Newly produced collagen fibrils could be observed along the cells in both groups I and II. This study demonstrated that a combination of fibroblast seeding and cyclic stretch could remodel and align the collagen fiber orientation in ECM-SIS bio-scaffolds. The better-aligned ECM-SIS has the prospect of eliciting improved effects on enhancing the healing of ligaments and tendons.

Bioreactor Design for Tendon/Ligament Engineering

“…Pneumatic actuators, [19][20][21] linear motors, 22,27 and step motorball screws (SMBSs) 2,42,76 are the most common actuators used in tendon/ligament bioreactors. Pneumatic actuators have several advantages, including the ease of maintenance, cleanliness, low cost, and high power-to-weight ratio.…”

“…35,42,76 In independent loading multichamber systems, linear motors are more popular due to their small size and relatively simple mechanical arrangement. 22,82 In the addition to the actuator, the connection between the mechanical input and the tissue is vital and often presents a major challenge. During dynamic loading of the tissue in culture, an even distribution of force throughout the entire sample is critical, otherwise tissue integrity is compromised by overloading the mechanical connection regions and/or by inhomogeneous mechanical stimulation.…”

“…Tensile force is focused on the side of constructs, which causes uneven distribution of mechanical stimulation. Although tissue clamps are the most popular and relatively effective method for holding tendon contructs, 22,42,82 the potential for damage at the clamping region needs to be considered. The method of reproducibly applying uniform loads to soft tissue without tissue damage or slippage is a critical problem in need of a satisfactory solution.…”

“…Therefore, the ideal design for the culture chamber should be similar. Like conventional cell culture, an unsealed chamber bioreactor connects to the outside environment through various ways, 19,22,27,35 such as the hydrophobic filter leak 42 and the labyrinth channel (Fig. 3).…”

“…In the past decade, promising results have been achieved using mesenchymal stem cells (MSCs) in tendon/ligament engineering. [16][17][18][19][20][21][22][23] However, the potential for MSCs to also differentiate into osteoblastic cells leading to ectopic bone formation raises concerns for their application in tendon repair. Likewise, bone marrow-derived MSCs that have been used in various studies [24][25][26][27][28] also pose a potential risk of ectopic bone formation.…”