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Electrohydrodynamic Printed Sub-microscale Fibrous Architectures Improved Cell Attachment and Collagen Type I Deposition
the fiber diameter ranging from 250 nm to 1 μm using an unprecedented opportunity to incorporate functional
electrospinning, which were found to facilitate attachment nanomaterials into the organized fibrous architectures
and osteogenic differentiation of human mesenchymal for enhanced bioactivity [21,22] . Especially for osteogenic
stem cells (hMSCs) in vitro. Ren et al. fabricated differentiation, nano hydroxyapatite (nHA) exhibits a
[9]
electrospinning nanofibers membranes with different strong componential similarity to native bones, which
ratios of PCL/gelatin for guided bone regeneration. has been proven to be good osteoconductive and
They found the fibrous membranes prompted calcium osteoinductive material [23,24] . Many existing studies have
deposition of MC3T3-E1 cells both in growth media and demonstrated that fibrous architectures with nHA can
osteogenic media. Nevertheless, the random deposition of facilitate osteogenic differentiation [25,26] . For example, Li
[27]
the micro/sub-microscale fibers increases the complexity et al. fabricated nHA/methacrylate gelatin/poly (l-lactic
of the fibrous structures, which makes it difficult to acid) electrospinning membranes with nHA concentration
decouple and decipher the interactions between the cells of 1% (nHA/solvent, w/v), which showed superior
and the ECM-mimetic fibers [10-12] . osteoinductivity compared to the membranes without
Electrohydrodynamic (EHD) printing is a newly nHA. However, few studies have realized organized sub-
emerging method for fabricating fibrous scaffolds with microscale fibrous architectures with nHA. In this study,
ultrafine fibers and user-defined organizations . Due to we investigated the effect of the scaffolds with microscale
[13]
its capability of depositing every single fiber in a highly and/or sub-microscale fibers on behaviors of MC3T3-E1
controlled manner, there has been an increasing interest in in vitro. The cellular attachment and spreading patterns
the investigation of cell-scaffold interactions using EHD were investigated by staining with vinculin and F-action.
printing [14-16] . For example, Brennan et al. EHD-printed The osteogenic differentiation of MC3T3-E1 on
[17]
PCL microscale fibrous scaffolds with a fiber diameter scaffolds with microscale and/or sub-microscale fibers,
of 4.01 μm and a fiber space ranging from 100 μm to and sub-microscale fibers with nHA was evaluated by
300 μm, and they found the scaffolds with the fiber space detecting collagen type I (COL-I) deposition and alkaline
of 100 μm enhanced collagen and mineral deposition of phosphatase (ALP) after 14 days of culture.
hMSCs. Eichholz et al. proposed microscale fibrous
[18]
scaffolds (fiber diameter of 10.4 μm) with four types of 2. Materials and methods
fiber orientation angles (90°, 45°, 10°, and random) using 2.1. Materials
EHD printing, which were further used to culture human
skeletal stem cells (hSSCs). Their results indicated Medical-grade PCL was bought from Jinan Daigang
that scaffolds with orthogonal architectures enhanced Biomaterial Co., Ltd (Mw = 80,000 g/mol, China).
osteogenesis of hSSCs via prompting yes-associated Polyethylene oxide (PEO) was bought from Aladdin Co.
protein nuclear translocation. Xie et al. investigated Ltd (Mw = 300,000 g/mol, Italy). nHA was bought from
[19]
the effect of fiber diameter (3 – 22 μm) on the spreading Aladdin Reagent Co., LTD (China). For solution-based
behaviors of bone marrow stem cells (BMSCs) and EHD printing of the sub-microscale fibrous architectures,
human umbilical vein endothelial cells. They found that PCL-PEO or PCL-PEO-nHA were dissolved or dispersed
the BMSCs (with a size of 200 μm) preferred to adhere in acetic acid solution, and the content of which was set
and bridge between thick fibers, while HUVECs (with a as 5% (w/v) for PCL, 6% (w/v) for PEO, and 0.5% (w/v)
size of 100 μm) can only adhere to the microfibers and for nHA, respectively.
form a circle to gradually fill the pore space. However, 2.2. Design and EHD printing of fibrous
the existing EHD-printed fibers are usually in microscale, scaffolds with micro/sub-microfibers
and few studies have been conducted to investigate the
response of cells on EHD-printed sub-microscale ECM- Three types of scaffolds with different structural and
mimetic fibers. compositional organizations were designed and fabricated
We previously developed a solution-based EHD using EHD printing techniques (Figure 1A-C). The
printing method for the fabrication of sub-microscale microscale fibrous scaffold (M) was designed with
fibrous architectures . The effect of sub-microscale a fiber spacing of 300 μm, a cross angle at 90°, and a
[20]
fibers (about 0.5 μm) on rat myocardial cells was fiber offset of 150 μm (Figure 1D). The micro/sub-
preliminarily investigated, with the results indicating that microscale fibrous scaffold (MS) was designed based on
sub-microscale fibers could enhance cellular adhesion the M scaffolds, which contained sub-microscale fibers
and orientation, whereas the effect of EHD-printed with a spacing of 100 μm. For fabrication, a total of five
sub-microscale fibrillar architectures on bone cells’ layers’ microscale fibers and five layers’ sub-microscale
adhesion patterns, spreading morphologies, growth, fibers were alternately stacked in a layer-by-layer manner
migration, and osteogenic differentiation was not clear. (Figure 1E). The sub-microscale fibrous scaffold with
Furthermore, solution-based EHD printing also provides nHA (MSN) shares the same structural organizations

2 International Journal of Bioprinting (2022)–Volume 8, Issue 2