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Hydrogel microfibers with perfusable folded channels for tissue constructs with folded morphology

Fiber-based materials with microchannels have drawn considerable attention in recent years owing to their ability to mimic intrinsic morphologies of living tissues. Folded morphologies, which are common in vivo, such as in skeletal muscle capillaries and intestine luminal endoderm, play important roles in the achievement of tissue functions. Here, microfibers with folded hollow channels are fabricated. Channel morphologies, such as straight-folded, double-folded and double-helical channels, can be regulated by adjusting flow conditions in the microfluidic devices. To further demonstrate the potential to be used in tissue engineering, intestine and skeletal muscle constructs are fabricated using these microfibers as building blocks. Furthermore, the properties of perfusability, permeability, cytocompatibility and weavability of the microfibers are evaluated. The asymmetric molecular distributions in the microfibers provide promising platforms for the study of nutrient exchange and energy supplement between normal and tortuous tissues. The new features of biofibers and proof-of-concept of tissue constructs with folded morphologies may contribute to the development of regenerative medicine and drug screening in the future.


Introduction
Fabricating tissue constructs in vitro can alleviate the crisis of organ shortage in transplantation, 1-3 and provide platforms for cancer research, 4 regenerative medicine and drug screening. 5,6 Mimicking three-dimensional tissue morphologies is a key point among processes of construction. 7 The folded morphology, which is common in vivo, such as in skeletal muscle capillaries, 8 the cerebral cortex, 9,10 intestine luminal endoderm and tracheal mucosa, [11][12][13][14] plays an important role in tissue functionalization, like oxygen ux improvement, information processing, nutrient absorption. Nevertheless, some folded morphologies cause diseases, such as asthma caused by folded mucosa penetrating into airway lumen. 15 In addition, blood vessels with folded channels, an anomalous morphology, are associated with various severe diseases like atherosclerosis, diabetes, hypertension, cancer and genetic diseases. [16][17][18][19][20][21][22][23] Thus, inherent biomechanical and geometrical mechanisms need to be studied for the purpose of developing new tools for the prevention and treatment of diseases. 18,24 Complexity of tissue structures makes it hard to build their folding morphologies. 25 At present, many efforts have been paid to construct folding structures in vitro, including by 3D bioprinting, electrospinning and microuidic techniques. [25][26][27][28][29][30][31][32] Current 3D bioprinting methods, based on layer-by-layer stacking, are usually difficult to print complex hollow structures, because layers must be connected and mechanically supported to avoid collapse and imprecise geometric congurations. 33 In the sacricial material-based method, platforms capable of fast bioink switching deposit sacricial materials into matrix material, occupying channels' positions at beginning and then dislodged aer molding. 34 As for electrospinning method, nanobers with several morphologies including porous, solid, core-shell, and hollow structures are produced. [35][36][37] However, the random whipping motion of the electrospun nanober makes it a challenge to handle single ber for constructing complex tissues. 38 As additional methods, microuidic techniques are developed to fabricate on-chip and ber-based tissue constructs. However, limited by conventional lithography methods, 39 channels embedded in PDMS chips usually have rectangle cross-sections, which are inconsistent with consecutive curved structures in vivo. In recent years, berbased techniques have been widely used in producing micro-bers with various morphologies, including solid, 40,41 helical, 42,43 grooved, 44 spindle, 45,46 folded shapes, 47 and the channel in microbers could be straight, 48 helical-channel and multi-channel. 49,50 Owing to the similarity to hierarchical structures in vivo, these microbers have the potential to be used as building blocks for tissue engineering. [51][52][53] Nevertheless, to the best of our knowledge, microbers with folded channels have not yet been reported.
In this study, we propose a microuidics-based method to fabricate microbers with double-folded, folded-straight and double-helical channels. Channel morphologies can be regulated by adjusting ow rates in the microuidic devices. Intestine and skeletal muscle constructs are fabricated using these microbers for future applications in tissue engineering. Moreover, characteristics of perfusability, permeability, biocompatibility and weavability endow these microbers with the potential to build up in vitro tortuous vessel models.

Fabrication of microbers
The microuidic device used to fabricate microbers was made up of two CaCl 2 inlets, one alginate inlet and one outlet. A 20 G blunt needle was punched as connector and alginate inlet. Two 32 G stainless needles were inserted into the connector in parallel as CaCl 2 inlets. A glass capillary (outer diameter, inner diameter and length are 1 mm, 0.75 mm and 2 cm respectively) was inserted from the other side serving as the outlet. All the connections were sealed with epoxy adhesive resin glue. Syringe pumps (Harvard PHD) were used to injecting the solutions into the microuidic device. The outlet of the device was immersed into CaCl 2 bath to form microbers.

Perfusion and permeation test of microbers
A home-made perfusion system was used in the perfusion experiment. The detailed information was introduced in our previous work. 49 To be brief, the microber was rstly sucked and xed into a glass capillary. Then uorescent particles solutions (diluted 500 times) were perfused into hollow channels through a three-way valve and recorded per 100 ms. For the permeation test, uorescent dextran solutions were perfused into hollow channels through a three-way valve. Aer dextran lling the channel, perfusion was stopped and the uorescence intensity was recorded per 10 s to 360 s and then per 60 s.

Cell culture
Human skeletal muscle cells (HSMCs, purchased from Shanghai and Shanghai Zhen Biotechnology Co., Ltd.) were maintained in DMEM supplemented with 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVEC, purchased from ScienCell) were maintained in complete ECM. Culture medium was changed every 2 days.

Cell staining
To evaluate cell viability, microbers with embedded cells were washed with PBS before stained with live/dead cell viability assay. Aer 20 minutes incubation, cells were released by dissolving the Ca-alginate matrix using 20 mM EDTA and imaged with confocal laser-scanning microscopy (Nikon, A1RSi). Soware Image J was used to count cells. To locate HSMCs in skeletal muscle constructs, HSMCs cultured on Petri dishes were rstly stained with 5 mM CellTracker Green in serum-free DMEM and incubated for 30 min. Then the labeled cells were dispensed into microbers at 5 Â 10 6 ml À1 and observed under microscope.

Characterization
The generation processes of microbers were recorded in realtime with the help of Full HD 1080p camera microscope. Bright eld and uorescence images were captured by Olympus IX81 microscopy. 3D uorescence images were obtained by Carl Zeiss LSM 780 microscopy. Scanning electron microscope (SEM, FEI Quanta 200) was used to illustrate channel positions. Threedimensional structure of microbers was snapped by Zeiss Lightsheet Z.1.

Generation of hollow hydrogel microbers with folded channels
The co-ow device designed for producing microbers with folded channels are illustrated in Fig. 1. Sodium alginate is chosen as sheath material while calcium chloride solution as core stream. Microbers with two hollow channels could soon be generated attributed to instantaneous crosslink reaction between alginate and calcium ion at the interface ( Fig. 1a and Video S1 in ESI †). Considering that the two aligned inner channels are equivalent in our experiment, microbers with double-folded channels (dfFiber, Fig. 1b), folded-straight channels (sfFiber, Fig. 1b), double-helical channels (dhFiber, Fig. 1b) and double-straight channels (dsFiber) are produced by adjusting the ow rate of core solution (Q i ), keeping sheath ow rates (Q o ) constant (200 ml min À1 ). To explain these phenomena, the relationship between ow patterns and Q i is illustrated in Fig. 1c. With Q i increasing, morphology of inner channels transforms from helical to folded and straight in turn. Demarcation lines of the four patterns vary according to device spec-ications and uid viscosity. Aside from the above-mentioned morphologies, numerous patterns can be realized by adding the number of embedded inner channels theoretically, such as microber with three-helical channels or three-folded channels (Fig. S1, Video S2 and S3 in ESI †). Moreover, the relationship between period (l) of df, dh, sf channels and ow rates are also explored. As demonstrated in Fig. 1d, under the circumstance that Q i /Q o is xed, raising the ow rates of Q i and Q o relates to decreased l.

Mechanism of microber generation
The formation mechanism has also been studied (Fig. 2). As mentioned earlier, inner stream tends to form straight channel when Q i /Q o is high, while helical channel is facilitated when Q i / Q o is low. The formation mechanism of helical channel can be explained by the hetero-generated liquid rope-coil effect. 49 To be brief, alginate solution nearby inner channels reacts with Ca 2+ rstly, forming a semi-crosslinked Ca-alginate layer with higher viscosity, which aerward causing the coiling phenomenon. However, in the case of sf-microber, the two streams are close with each other and thus uid-ows are not independent (Fig. 2a). The thin layer of alginate sandwiched between the two streams is fully crosslinked owing to rapid and thoroughly Ca 2+ diffusion (Fig. 2d). Therefore, the coiling trend is inuenced by the nearby straight stream and the resultant ow pattern becomes a vibrating folded channel paralleling to a straight one. We dene this mechanism as "gelation-induced guiding effect". The straight and sine-wave channels serve as "guiding channel" and "conned channel" respectively. This explanation suits for the formation of dfFibers and three-folded bers as well. To conrm this speculation, microbers are injected into water instead of CaCl 2 solution in order to diffuse uncrosslinked alginate (Fig. 2b and c). The gelation part is clearly demonstrated in the scanning electron microscope images.

Intestine construct fabricated using microbers
The new features of microbers enable their potential in fabricating tissue constructs with folded morphologies. Folding morphologies can be found commonly in tissues, such as small intestine. Coiling sfFibers with folded channels inward, small intestine constructs are fabricated in vitro (Fig. 3). Side and cross-section view of intestine constructs are presented in Fig. 3d, e and b respectively. Immersed in CaCl 2 solution to crosslink microbers, a complete intestine construct is built and the ability of being raised by tweezers indicates enough strength for manipulation, as shown in Fig. 3c. This may provide a simple intestine tissue construct for tissue engineering.

Skeletal muscle constructs fabricated using microbers
Apart from intestine constructs, human skeletal muscle ber is constructed (Fig. 4 and Video S4 in ESI †). In contracted state, muscle bers are positioned into the polygonal regions encircled by convoluted capillaries and anastomotic vessels. Using our microber-based strategy, capillaries between fascicles are structurally simulated by folded channels. Thus a muscle fascicle unit is established by embedding one straight micro-ber into several surrounded dfFibers (Fig. 4b and c). In addition, human skeletal muscle cells (HSMCs) are cultured in the  fascicle unit in order to test the biocompatibility (Fig. 5a). Live/ dead staining results reveal that high viability (over 90%) still remains aer 7 days culture indicating that this construct possesses the possibility for tissue engineering (Fig. 5b).

Properties of microbers with folded channels
Moreover, properties of perfusability, permeability, biocompatibility and weavability of the microber have been investigated (Fig. 6). In the permeability test, FITC-dextran (green, 70 kDa), TRITC-dextran (red, 70 kDa) are dissolved in CaCl 2 and perfused in the sfFiber simultaneously (Fig. 6a). The uorescent intensity of two square areas with same distance to the two inner channels and three lines across the microber is measured. The relationship between relative uorescent intensity and time is depicted in Fig. 6b. The uorescent intensity increases rapidly in the beginning and then slowly decreases. The diffuse rate of the folded channel is calculated to be 2.83 times greater than the straight channel (ESI †), presenting faster material exchange capacity. The possible explanation of this phenomenon is that the channel with folded morphology has a higher surface area than straight channel in same length of microber. An asymmetric concentration distribution is observed by folded channel in microber due to its asymmetric position. The c side has the same concentration gradient, while the d side differs about 2.71 times (Fig. 6c, ESI †). This phenomenon could be explained that the uorescent molecules diffuse faster in outer solution than in microbers. The distance between folded channel and the edge of microber is near at line 1 position and far at line 3 position from d side, thus the different concentration gradient is observed.
Furthermore, perfusability of microbers is studied using dfFiber ( Fig. 6d and Video S5 † in ESI †). Green and red uorescence particles are perfused through two channels separately, and their ow rates can be independent. Moreover, the biocompatibility has also been studied (Fig. 6e). Human umbilical vein endothelial cells (HUVECs) are embedded in microbers and cultured at 37 C and viability data are collected every 12 hours. As shown in Fig. 6e, the viability of embedded HUVECs is 92% aer 48 hours culture, indicating that the proposed method holds great biocompatibility. In addition, weavability of microbers with folded channels is explored by knotting (Fig. S2, ESI †). Moreover, microbers with folded channels may provide platforms for pathological study. For instance, the physiological environment of blood vessel, such as vessel tortuosity, blood pressure, viscosity and shear force, plays a vital role in the pathology of atherosclerosis. Despite tremendous efforts have been paid, the interplay between them remains unclear. 54 The models proposed in this work can be used as platforms for the evaluation of endothelial cell responses to atherogenic factors, including ROS and lipoprotein permeability, under specic blood and vessel tortuosity, which benets further researches on the prevention of   atherosclerosis. This model may also be used in elds of drug screening and pathophysiology, exploring the difference of drug diffusion, materials exchange and energy supplement between normal and tortuous tissues in future studies.

Conclusions
In summary, we propose a microuidics-based method to fabricate microbers with folded hollow channels, which can be used as building blocks for tissue constructs in vitro. By adjusting the ow rates, microbers with various channel morphologies including double-folded, folded-straight and double-helical are produced. The gelation-induced guiding effect is used to understand the intrinsic mechanism. The proof-of-concept in vitro intestine and skeletal muscle constructs are produced using the above-mentioned micro-bers. Moreover, characteristics of perfusability, permeability and asymmetric molecular distributions in microbers make it possible to be used as platforms for the study of nutrient exchange and energy supplement between normal and tortuous tissues.

Conflicts of interest
There are no conicts to declare.