1. Introduction
Tubular systems are responsible for diverse physiological and pathological processes related to all organs
in vivo and for the survival of engineered tissues
in vitro [
1], [
2]. Such systems, which range from microtubule-associated proteins to circulatory and respiratory components, play key roles in the transportation of oxygen and nutrients and the removal of waste products [
3], [
4]. Significant studies have shown that perfusable tubular structures in engineered tissues can enhance the function and/or ingrowth of blood vessels from the host [
5], [
6], [
7]. To realize the fabrication of such tubular systems, various technologies have emerged, such as micro-molding [
8], [
9], three-dimensional (3D) printing [
9], [
10], [
11], and electro-spinning [
12], providing great opportunities for medical applications [
13], [
14], [
15], [
16]. Although there have been many successes, most studies based on these technologies focus on the fabrication of tubular structures with simple and smooth layers, making it difficult to reproduce the much more complex anatomical structures of actual native tubular tissues and thereby limiting the development of biomimetic tissue engineering. Thus, there is still a great need for a new method to fabricate heterogeneous tubes with complex biomimetic structures and biological features.
In this paper, we present a novel one-step microfluidic spiraling-spinning stratagem for the continuous fabrication of conduits with biomimetic helix constructions, as shown in the schematic in
Fig. 1. Microfluidics is a revolutionary platform for producing micro- or nano-scale structures with diverse shapes [
17], [
18], [
19], [
20], [
21]. Thanks to their moderate reaction conditions and a wide arrangement of available biomaterials, microfluidics systems have recently received great attention for fabricating microfibers with variant shapes and sizes [
22], [
23]. In particular, microfibers with desirable uniformity and adjustable geometries—including hollow [
24], [
25], grooved [
26], [
27], flat [
28], and even helical fibers [
29], [
30]—have been successfully produced based on specific microfluidic platforms. Moreover, by simply tuning the operating parameters of the microfluidics, such as the chemical composition and flowrate, continuous spatiotemporally coded fibers with complex morphologies and regulatory constituents can be obtained [
31], [
32], [
33], [
34], [
35], [
36], [
37]. However, it is still challenging to generate conduits with a screwed surface using microfluidics, and the underlying value of screwed conduits (SCs) for biomedical applications remains unexplored.
In this research, we employed a simple coaxial microfluidic device to realize the scalable generation of the desired bio-inspired screwed conduits (BSCs) via the microfluidic rope-coiling effect for the purpose of constructing microvessels and bronchioles. The inner phase of the microfluidics is a mixture of ultraviolet (UV)- and ion-curable precursors, while the outer phase and collecting base are a chemically inert aqueous solution and a calcium ion solution, respectively. When the ratio of the inner/outer flow rates is increased, the consecutive precursor’s stream forms helical structures and is even closely packed into a tubular structure in the microchannel due to the rope-coiling effect. Thus, via the fast crosslinking of these precursors, SCs with regular spiral structures are continuously generated. By taking advantage of the designable injection microchannel of microfluidics, BSCs with diverse structures—such as Janus, triplex, core-shell, and even multilayered structures—can be created. Through a fluid perfusion study and permeation kinetics studies, we demonstrate that the screwed hydrogel conduits have good perfusability and permeability for various biomolecules. This advantage, together with the specific morphology of BSCs, allows various cells to form functional physiological structures on it, such as human umbilical vein endothelial cells (HUVECs), human pulmonary alveolar epithelial cells (HPAs), and myogenic cells (MCs). These results indicate that the hydrogel BSCs hold potential for use as templates for the creation of artificial bronchioles and microvessels in tissue engineering and regenerative medicine.
2. Results and discussion
In a previous study [
33], we produced alginate fibers with specific spatial and compositional components, along with straight, bead-like, and helical-fiber structures, in a typical continuous microfluidic device (Fig. S1(a) in Appendix A). In the current research, an inner phase of high-viscosity sodium alginate (Na-Alg) mixed with polyethylene glycol diacrylate (PEGDA) precursors was pumped into the inner capillary in the same direction as the square tube. To maintain the structure of the inner stream and prevent diffusion of the alginate, a polyethylene glycol (PEG) solution was adopted as an external low-viscosity phase. When the flowrate set reached a certain value, the viscous stream jet from the orifice first curved into a helical shape due to the rope-coiling effect in the microchannel. With the constant resistance from the fluid, the continual helical stream generated by the precursor was closely packed into a tubular structure along the channel, as shown in
Fig. 2(a). As the packed helical stream formed into a conduit that ran along the collection channel, we introduced UV light to immediately crosslink one component of the hydrogel (PEGDA) before the hydrogel conduit jetted into the collection base, in what we term “semi-crosslinking.” Then, the calcium ions from the collection base diffused into the packed semi-crosslinked helical fiber, facilitating the complete gelation of the hydrogel precursor. In this way, BSCs were continuously generated in the collection container (
Fig. 2(b); Fig. S1 in Appendix A). The surface of the obtained conduits has a regular spiral pattern, and the conduits are soft and feature uniform walls (
Fig. 2(c); Fig. S1(b)). Uniform folds, as well as hollow cross-sectional structures, can be observed in the scanning electron micrographs (
Fig. 2(c-iii); Fig. S1(c)). The perfusability and permeability of the BSCs were also examined (Figs. S2 and S3 in Appendix A). Notably, the velocity sets and viscosity of the inner jets greatly affected the running of the microfluidic fabrication process, such that different flowrate sets and precursor concentrations formed BSCs with different structures (
Figs. 2(d) and (e); Figs. S1(c), S4, and S5 in Appendix A).
By using the flexibility and feasibility of microfluidics for constructing tubes with heterogeneous microfiber modules, we discovered that the SCs could be endowed with multiple components as well (
Fig. 3). To fabricate screwed hydrogel conduits with the desired multiple components, we used a conical double-barrel capillary to serve as the injecting channel, which was coaxially inserted into the collecting capillary. Two pre-gel solutions with different fluorescent polystyrene nanoparticles representing different components were then synchronously pumped into the injecting capillary channel, as depicted in
Fig. 3(a-i). Because of the higher surface tension effects and the micrometer scale size of the microchannel, the fluids existed in the form of laminar flow within the channel, and diffusion was rare between the interface of the different fluids during the movement of the microfibers. As a result, the generated micro-fibrous modules exhibited the same heterogeneous structure and composition distribution as the injecting stream. We discovered that the stream of ejecting fluids with multiple components also formed a coiling sequence, like a single ejecting fluid [
19]. Therefore, helical streams with a Janus structure were formed first, and conduits with an alternate Janus composition were subsequently achieved (
Fig. 3(b-i)).
Confocal laser scanning microscopy (CLSM) of the longitudinal section of the hydrogel conduits (
Fig. 3(c-i)) showed that the tubular structure was steady and the stream was tightly packed. The inner construction of the modular microfiber of the conduits exhibited a viewable boundary between the two compositions, indicating that little mass diffusion had occurred between them during the conduit fabrication. By replacing the dual-barrel injecting capillary with a three-barrel one, triplex-component screwed hydrogel conduits with various chemical components were fabricated as well (Figs. 3(b-ii) and (c-ii)). Thus, multiple component grooved microtubes were made, with underlying for a variety of applications in the creation of multi-active encapsulations (Figs. S6 and S7(a) in Appendix A).
In addition to mono-hierarchical grooved tubular microfibers with various compositions, it was possible to fabricate multilayered microstructure tubes through a simple transformation of the microfluidics device involving hierarchical injecting channels, as shown in Figs. 3(a-iii) and (a-iv). A core fluid of Na-Alg solution with green fluorescent particles, a middle fluid of Na-Alg solution with red fluorescent particles, and a sheath fluid of PEG solution were synchronously injected into the corresponding channels in the desired flow patterns (
Fig. 3(a-iii)). A coaxial flow formed first, while the core flow came into contact with the middle flow, and then the coaxial flow was packed into the grooved conduits in the square channel. The tubular microfiber underwent the rapid diffusion of Ca
2+ ions in the collection base, which improved the gelation, and finally stacked spirally in the collecting channel to form a helical morphology. Following the persistent gelation, solidified conduits with a hierarchical double-layered structure were continuously obtained (Figs. 3(b-iii) and (c-iii)). The double-layered modular conduits had a perfect spiral groove, similar to the simple or heterogeneous conduits fabricated earlier. The preparation of BSCs with tri-laminar isotropic modules was achieved by means of the coaxial assembly of three capillaries as the hierarchical injection channel (
Fig. 3(a-iv)). After the movement of the fluid and the completed crosslinking reaction, conduits with a triple-layer architecture were obtained (Figs. 3(b-iv) and (c-iv)). Nevertheless, since the instantaneous flowrate could not match the gelation velocity of the fluid in different positions, the thicknesses of the layers were slightly different in the microfluidic channel. Therefore, the generation of hierarchical conduits with a uniform and regular shell was complicated (Figs. S7(b) and S8 in Appendix A).
Such multicomponent micro-structured tubes offer versatile modulation of chemical and morphological properties at the micron scale. The encapsulation of different materials at the micron scale may offer new opportunities to generate a range of useful functions in materials. Nature also uses the spatial patterning of existing materials to generate new functions. Our proposed approach can thus be used to fabricate many other materials in a spatially modulated mode to produce functional tubes with structures conducive to a diverse range of applications, including tissue engineering and drug delivery.
To identify the potential of the BSCs for the construction of tubular tissue for tissue engineering, bio-inspired vessels and bronchioles were constructed based on the BSCs. First, we dispersed gelatin acryloyl (GelMA) into an alginate aqueous solution to replace the UV-curable component PEG, because GelMA is known to be a bioactive hydrogel with cytocompatibility. The use of composite alginate-GelMA as the material of the hydrogel scaffolds could better support cell adhesion and growth compared with alginate alone. HUVECs were adopted as typical seed cells for the construction of bionic microvessels; the manufacturing processes are shown in
Fig. 4(a). With the HUVECs pumped into the BSC scaffolds, they were combined as a complex (Fig. S9 in Appendix A). In particular, the BSC scaffolds provided a 3D microenvironment for the HUVECs’ growth, where the endothelial cells (ECs) were uniformly distributed inside (Fig. S10 in Appendix A). After culturing for days, cell proliferation occurred in the BSC scaffolds (
Fig. 4(b); Figs. S10 and S11 in Appendix A). Because induction of the formation of confluent monolayers of HUVECs is crucial for the evaluation of mimetic scaffolded vessels, the formation of cell layers was studied using immunofluorescence (Fig. S12 in Appendix A). From the CLSM images, it was observed that the initially homogeneously dispersed ECs migrated toward the peripheries of the inner face of the hydrogel conduits after seven days of culture. Meanwhile, the HUVECs formed a uniform layer inside the inner face of the hydrogel conduits, with the lumen structure of the HUVECs being observable from the section view (
Fig. 4(c)). In addition, images from F-actin staining revealed that the ECs were organized into intact cell monolayers in the BSC scaffold. Immunofluorescence staining for cluster of differentiation 31 (CD31) was also performed to verify the existence of tight junctions between vascular HUVECs (
Fig. 4(d); Fig. S13 in Appendix A). The dispersed expression of CD31 demonstrated a homogeneous cell layer maintained by HUVECs in the BSC.
The generation of bionic bronchioles was similar to that of the bionic vessels described above; the HPAs suspension was instilled into the BSC as demonstrated in
Fig. 4(e). HPAs monolayers proliferated in the BSC and formed tubular tissues with lumen structures after seven days of culture (
Figs. 4(f) and (g)). Similar to the formation of the vessels, the formation of an induced cell confluent monolayer is critical for the evaluation of bionic bronchi, so the formation of cell layers was also studied using immunofluorescence. F-actin staining indicated that intact HPAs monolayers had formed on the inner wall of the BSC. The results demonstrated that the BSC scaffolds also possess the ability to promote the proliferation and epithelization of HPAs. Next, immunofluorescence staining for E-cadherin was performed to verify the existence of tight junctions between the lung epithelial cells (
Fig. 4(h); Fig. S14 in Appendix A). The uniformly dispersed expression of E-cadherin demonstrated the existence of a homogeneous cell layer generated by HPAs in the BSC scaffold. The experimental results demonstrated that the BSC scaffold not only provided an architecture that allowed HPAs to proliferate and differentiate but also enabled a 3D environment for the adhesion, growth, and epithelialization of HPAs. Therefore, our study may afford a new
in vitro model for exploring the function variation of blood vessels and bronchioles with the alteration of structures, perfusability, and permeabilities, further acting as a novel platform for biological research
in vitro.
In addition, we explored the potential of BSCs with special structures and morphologies for inducing cell differentiation. We examined the possibility of using the BSCs as a novel scaffold for inducing MCs to differentiate into a helical muscular bundle by encapsulating C2C12 cells inside conduits. In the fabrication process, as shown in
Fig. 5(a), a coaxial device was chosen to generate the cell-laden BSC scaffolds. Red fluorescent-labeled C2C12 cells suspended in a fibrinogen aqueous solution served as the cell stream. The cell stream was then encapsulated in the core of the BSCs, which was followed by the coiling of the fibers (Fig. S15 in Appendix A). After being cultured in an incubator for several days, a coiling muscular bundle embedded in the tube was formed. The CLSM image showed that the obtained muscular bundles possessed a stable pitch with tightly connected cells benefitting from the structure of the BSCs (
Fig. 5(b)). In addition, the F-actin dye images indicated that the myoblasts had differentiated into myotubes, which proved that the spatial structure of the BSCs could induce the differentiation and proliferation of C2C12 cells (
Fig. 5(c)). The results indicated that we were able to achieve MCs encapsulation by encapsulating the cells
in situ, as well as subsequent effects on MCs proliferation and induction through the unique structural morphology of the BSCs. These properties indicate further potential for the use of BSCs in the reconstitution of encircling tubular muscle tissue.
The special surface morphologies of the BSCs for cell differentiation were also examined. Unlike the abovementioned construction of vessels, two essential factors were necessary in order to mimic structural vessels: namely, organizing vascular endothelium inside the scaffolds and directing the aligned growth of MCs circumferentially outside the scaffolds. Here, we selected HUVECs and C2C12 cells as typical ECs and MCs, respectively. The biomimetic vessel was built as shown in
Fig. 5(d), and C2C12 cells were first inoculated on the external surface of the BSCs due to their slower growth rate. After the density of C2C12 cells reached 80%, HUVECs were further seeded in the internal surface of the BSCs to form the composite (
Fig. 5(e) and S16 in Appendix A). As observed in the CLSM images and cross-sectional images, HUVECs grew densely on the internal channel of the BSCs, forming a cellular monolayer (
Figs. 5(f) and (g); Fig. S17 in Appendix A).
The orientation of the C2C12 cells in the tubular microfibers was also examined. The angle between the long-axis direction of the cells and the circumferential direction of the groove was determined (
Fig. 5(h)). In general, the measured angles ranged from 0° to 90°, and numbers were counted for every 10°. The results indicated that over 60% of the C2C12 cells exhibited an aspect within 30° in the scaffolds. Moreover, the ECs and MCs were distributed on the inner and outer surfaces of the BSCs, respectively, resembling the intima and media layer of a vessel. These characteristics of the BSCs make them a perfect tool to construct bio-inspired vascular tissue with circumferentially aligned MCs and monolayered ECs for the mimicry of real vessels
in vivo. Furthermore, the microfibers loaded with cells exhibited feasible efficacy for potential application in the areas of organs-on-a-chip and tissue engineering. Thus, the designed screwed hydrogel conduits could have remarkable feasibility when applied in biomedical fields including blood-vessels-on-a-chip and bronchioles-on-a-chip.
3. Conclusions
In this work, we presented a novel approach for the consecutive spinning of BSCs through a classical coaxial microfluidic device. Parameters such as the length, diameter, and pitch of the helical grooves were accurately controlled via regulation of the flowrates and concentration of the phase stream. Fibrous modules of the grooved conduits with new Janus, triplex, and core-shell structures were fabricated by varying the components of the core flow phase in the microfluidic device. A fluid perfusion study and permeation kinetic studies demonstrated that the screwed hydrogel conduits exhibited favorable perfusability and permeability for various biomolecules. Taking advantage of these attractive properties, including the unique morphological characteristics, excellent perfusability, and good permeability, the use of the SCs to mimic blood vessels and bronchioles was demonstrated. The results indicated that such grooved hydrogel conduits are highly versatile for tissue engineering applications. Therefore, we believe that various applications of these BSCs based on the microfluidic rope-coiling effect will be discovered in diverse areas.
Acknowledgments
This work was supported by the National Key Research and Development Program of China (2020YFA0710800), the Key Program of National Natural Science Foundation of China (81930043 and 82330055), and the National Natural Science Foundation of China (82101184).
Compliance with ethics guidelines
Rui Liu, Jiahui Guo, Bin Kong, Yunru Yu, Yuanjin Zhao, and Lingyun Sun declare that they have no conflict of interest or financial conflicts to disclose.
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