Bi‐Layered Tubular Microfiber Scaffolds as Functional Templates for Engineering Human Intestinal Smooth Muscle Tissue

Abstract

Designing biomimetic scaffolds with in vivo-like microenvironments using biomaterials is an essential component of successful tissue engineering approaches. The intestinal smooth muscle layers exhibit a complex tubular structure consisting of two concentric muscle layers in which the inner circular layer is orthogonally oriented to the outer longitudinal layer. Here, a 3D bi-layered tubular scaffold is presented based on flexible, mechanically robust, and well aligned silk protein microfibers to mimic the native human intestinal smooth muscle structure. The scaffolds are seeded with primary human intestinal smooth muscle cells to replicate intestinal muscle tissues in vitro. Characterization of the tissue constructs reveals good biocompatibility and support for cell alignment and elongation in the different scaffold layers to enhance cell differentiation and functions. Furthermore, the engineered smooth muscle constructs support oriented neurite outgrowth, a requisite step to achieve functional innervation. These results suggest these microfiber scaffolds as functional templates for in vitro regeneration of human intestinal smooth muscle systems. The scaffolding provides a crucial step toward engineering functional human intestinal tissue in vitro, as well as engineering other types of smooth muscles in terms of their similar phenotypes. Such utility may lead to a better understanding of smooth muscle associated diseases and treatments.

Engineering Complex Anisotropic Scaffolds beyond Simply Uniaxial Alignment for Tissue Engineering

“…The manual rolling assembly is mainly developed to produce 3D scaffolds with TMA by warping around a mandrel (Table 3D), such as single-step rolling of stacked two strips with opposing ±30° alignment [78][79][80] or one strip having alternating ±30° patterned regions [102] into an angle-ply multilamellar scaffold for annulus fibrosus, and orthogonal rolling of sheets into bilayered scaffolds for smooth muscle. [13] During this rolling process, the tubular dimensions including outer diameter, inner diameter, and height can be regulated easily by changing the mandrel diameter, the number of wrapped lamellas and the width of 2D aligned building blocks, respectively. Additionally, the rolling assembly also has demonstrated the ability to create directly the columnar scaffolds, [10,65] and transform into nanofibrous bundles [69] or small tubes [66] as 1D aligned building blocks for engineering the HUA without a mandrel (Table 3D).…”

“…Providing sufficient mechanical strength for loadbearing function, guiding AF cell oriented growth and ECM deposition, and supporting differentiation of mesenchymal stem cells (MSCs) to chondrogenic lineage [77][78][79][80]93,[96][97]102,172] Smooth muscle in intestines Guiding organization of SMCs similar to that of circular and longitudinal smooth muscle [13,81,82] Blood vessel Recovering the SMCs and ECs into in vivo morphology [83,101,135,193,194] As mentioned above, skeletal muscle is a typical HUA tissue composed of long and thick myofibers that fused and differentiated from myoblasts, thus the scaffolds for skeletal muscle have been developed for mimicking the HUA architecture to present critical topographical cues for prealignment of the myoblasts, guiding the myoblasts fusion, promoting myotube formation and possible maturation into functional myofibers, and maintaining the hierarchical arrangement of myotubes, aiming to regenerate the structure and function of volumetric muscle defects.…”

“…Indeed, various fabrication approaches, such as micropattern, freeze-drying, and electrospinning, have so far been explored to confer aligned features to scaffolds for anisotropic tissue engineering. [5,[7][8][9] Although the anisotropic similarity between these scaffolds and native tissues is often emphasized, one readily notices that there still is a big difference in their anisotropic complexity for most native tissues, [10][11][12][13][14] inducing these scaffolds to recapitulate incompletely the native form and function of tissue organization. Most of the methods listed above only can produce the scaffolds with simply, homogeneously uniaxial alignment.…”

Tissue engineering of the gastrointestinal tract: the historic path to translation

“…Differentiated functional neurons were shown through real time force generation and staining showed innervation of SMCs [ 75 , 130 – 132 ]. Finally, a 3D bi-layered silk protein scaffold has been utilized as a support ECM for intestinal smooth muscle cells and have been shown to support cell function, differentiation, and neurite growth [ 133 ].…”

A Bioprinted Tubular Intestine Model Using a Colon‐Specific Extracellular Matrix Bioink

“…For example, the 3D geometry of intestinal villi can be printed using an extrusion-based printing system. [93] In addition, a tubular scaffold can provide 3D geometries for cell accommodation [12,14] or a tubular architecture could be spontaneously generated by cellular self-organization after printing. [15] It has been previously reported that such a hollow tubular structure provides distinctive advantages in intestine models: a physiological oxygen gradient can be readily created across the transmural interface, keeping primary epithelial cells viable and properly polarized while providing a platform to coculture anaerobes.…”