Fabrication of single and bundled filament-like tissues using biodegradable hyaluronic acid-based hollow hydrogel fibers

https://doi.org/10.1016/j.ijbiomac.2017.06.013Get rights and content

Abstract

Hydrogel fibers with biodegradable and biocompatible features are useful for the fabrication of filament-like tissues. We developed cell-laden hyaluronic acid (HA)-based hollow hydrogel fibers to create single and bundled filament-like tissues. The cell-laden fibers were fabricated by crosslinking phenolic-substituted hyaluronic acid (HA-Ph) in an aqueous solution containing cells through a horseradish peroxidase (HRP)-catalyzed reaction in the presence of catalase by extruding the solution in ambient flow of an aqueous solution containing H2O2. The encapsulated cells proliferated and grew within the hollow core, and the cells formed filament-like constructs in both single and bundled fibers, which were obtained by collection on a rotating cylindrical tube. Single and bundled filament-like tissues covered with an additional heterogeneous cell layer were obtained by degrading the fiber membrane using hyaluronidase after covering the fiber surface with heterogeneous cells. Cellular viability was preserved during HA-Ph hydrogel fiber fabrication and filament-like tissue formation. These results demonstrate the feasibility of HA-based hollow hydrogel fibers obtained through HRP- and catalase-mediated reactions to engineer filament-like tissues.

Graphical abstract

Fabrication of single and bundled filament-like tissues using hyaluronic acid-based hollow hydrogel fibers.

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Introduction

Hydrogels have emerged as leading matrices for engineered tissue scaffolds because of their hydrophilic characteristics and potential for biocompatibility [1], [2], [3], [4], [5]. Among hydrogels, extracellular matrix (ECM)-derived components have been recognized as ideal templates to prepare cell-laden hydrogels that can mimic the structural and biological properties of cellular environments in vivo [2], [4], [5], [6]. In particular, hyaluronic acid (HA), a major ECM component in various tissues, is an attractive template for the construction of hydrogels with desired morphology, mechanical strength, and bioactivity [2], [7], [8]. Until now, HA-based hydrogels have been used for tissue engineering applications in various shapes and architectures such as sheets, bulk constructs, microparticles, and microcapsules [9], [10], [11], [12], [13]. These hydrogels provide stable three-dimensional cultivation systems for the survival, expansion, and self-renewal of cells as well as differentiation of encapsulated cells [9], [11], [12], [13]. Among the various cell-laden hydrogels, fiber-shaped vehicles can be advantageous for fabrication of tissue-like biological constructs that mimic the structural and physical properties of native tissues [1], [3], [4], [5], [6]. In addition, fibers can be processed into woven, non-woven, and knitted constructs to fabricate complex tissue-like biological constructs [3], [4], [5], [6]. To the best of our knowledge, no report has adopted an HA-based hydrogel as a fiber-shaped vehicle for cell encapsulation.

In this study, we have developed an HA-based hydrogel as a tool to fabricate a cell-laden hollow fiber-shaped vehicle. Cell-laden hydrogel fibers have commonly been fabricated from unique or composite polymers, including gelatin, collagen, alginate, amylopectin, and chitosan [1], [3], [4], [5], [6], [14], [15], through techniques such as electrospinning, microfluidic spinning, wetspinning, and interfacial complexation [1], [3], [4], [5], [6], [13], [14], [15], [16], [17], [18]. Among the various existing fabrication methods, the microfluidic spinning technique allows precise control over the size, geometry, morphology, and chemical features of fibers [1], [3], [4], [5], [6], [13], [14], [15], [16], [17], [18]. Previously, we produced alginate-based hollow hydrogel fibers through enzymatic crosslinking and degradation using a coaxial double-orifice spinneret [14]. In this system, horseradish peroxidase (HRP) and catalase, both of which consume H2O2, were used to form a hollow in the hydrogel vehicles [14], [19], [20]. To this end, phenolic-substituted HA (HA-Ph) may be a useful bioactive and biodegradable material to create hollow hydrogel fibers that enable cell encapsulation while maintaining cell viability [9], [12], [21]. The HA-Ph fibers were made by a microfluidic spinning technique in which an aqueous solution of HA-Ph containing HRP and catalase was extruded into an ambient co-flowing aqueous solution containing H2O2 through a coaxial double-orifice spinneret. Subsequently, the fabricated fibers were simply bundled by rolling on a rotating cylindrical tube to prepare a higher-order fiber-shaped construct (Fig. 1B and C). The attractive point of using single and bundled HA-Ph hollow hydrogel fibers is that they provide a biomimetic environment for the growth of encapsulated cells while maintaining biodegradability.

In this study, we demonstrated the feasibility of single and bundled filament-like tissue fabrication using HA-Ph fiber-shaped hydrogel vehicles. We first investigated the diameter and membrane thickness of HA-Ph hydrogel fibers affected by changes in both the velocities of extruded solutions in the coaxial double-orifice spinneret and the concentration of reactants. Next, we examined the cytotoxicity of the fiber fabrication process and growth profiles of encapsulated cells in the HA-Ph hydrogel fibers. Finally, surface-coating with cells and the subsequent degradation of the HA-Ph hydrogel fibers were investigated to create a filament-like tissue construct covered with a heterogeneous cell layer. This study revealed the feasibility of HA-Ph cell-laden fiber-shaped vehicles for tissue engineering applications.

Section snippets

Materials

Sodium-HA (average molecular weight: 1.7 × 106 Da) was obtained from JNC Corp. (Tokyo, Japan). HRP (160 U/mg), H2O2 aqueous solution [30% (w/w)] and catalase (1.3 × 104 U/mg, from bovine liver) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Gelatin (type B; from porcine skin, 300 Bloom) and hyaluronidase from sheep testes (type II, ≥300 U/mg) were purchased from Sigma (St. Louis, MO, USA). HA-Ph and phenolic-substituted gelatin (gelatin-Ph) were prepared according to previously

Results and discussion

Hydrogels of HA-Ph obtained through an HRP-mediated reaction have been investigated as a promising biomaterial for various biomedical applications such as drug delivery, tissue engineering, and peritoneal adhesion prevention because of their excellent biocompatibility and biodegradability [8], [9], [10], [12], [22]. In addition, HA-Ph hydrogels have been used for encapsulation of cells in a variety of shapes and architectures [9], [12]. These studies illustrate that HA-Ph hydrogel is an

Conclusion

The current study reports the preparation of single and bundled cell-laden HA-Ph hollow hydrogel fibers as a template for filament-like tissues in tissue engineering applications. The HA-Ph hydrogel fibers were fabricated using a coaxial double-orifice spinneret through HRP- and catalase-mediated reactions. The fabricated cell-laden HA-Ph hydrogel fibers were bundled by rolling on a rotating cylindrical plastic tube to obtain a macro-scale fiber-shaped construct. The diameter and membrane

Acknowledgement

This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Numbers 15H04194 and 16H02423.

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