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International Journal of Bioprinting Osteogenic differentiation of hMSCs by PBF-LB

biology. The putative factors that play key roles in stem cell hMSCs, in association with unidirectional bone matrix
differentiation have been believed biochemical molecules, organization.
including growth factors and biological supplements.

However, it is increasingly recognized that environmental 2. Materials and methods
cues can determine cell fate specifications. Limited reports 2.1. Fabrication of substrates by metal additive
4
have shown that stem cell lineage can be controlled using manufacturing
only inherent material properties. For example, scaffold The substrates were fabricated from Ti-6Al-4V powder us-
mechanical stiffness, surface topography, and materials ing PBF-LB based on the computer-aided design (CAD)
5,6
7,8
degradation 9,10 have been reported to impact stem cell fate. data. The substrates were then acid-cleaned with nitric
Topographical control of somatic cells has been developed hydrofluoric acid to remove molding defects that occur
over decades and is associated with nanotechnological during the layered molding process. After acid cleaning
advances. 11-13 Metal additive manufacturing is a powerful was completed, the substrate was degreased, and impuri-
tool for providing the desired material properties through ties were removed from the surface by ultrasonic cleaning.
three-dimensional (3D) structural design. 14,15 This The substrates were sterilized (121°C, 20 min) in an auto-
technique realizes the fabrication of required structure with clave (Series 540, NAPCO) for use in cell culture tests.
particular shape and porosity from micrometer-sized metal
powders, which serve as the starting materials. Focusing 2.2. Analysis of substrate surface topography
on the microstructure of intact bone tissue, orderly- A confocal laser microscope (VK-9700, Keyence, Osaka,
shaped materials mimicking the bone matrix structure Japan) was used to quantitatively evaluate the surface
have serious advantages for guiding functional bone topography of the substrates. Line profiles were acquired
regeneration. 16-19 Living bone tissue exhibits high-strength per field of view, and the peak width and height were
mechanical characteristics in the required direction measured for each profile. For the control substrate, the
because of preferred collagen elongation and apatite crystal surface roughness (Sa; arithmetical mean height), which
formation. The crystallographic anisotropy of bone varies means area roughness, was measured. In addition, the
20
21
significantly depending on the anatomical portion. surface topography of the substrate was observed by
However, it is difficult to spontaneously reconstruct bone field-emission scanning electron microscopy (FE-SEM;
matrix microstructure in regenerated or diseased bones JIB-4610F, JEOL, Japan) operated at 20 kV and 16 mA.
that have lost their appropriate organized matrix, even Substrates were washed with acid for a short time, followed
if the bone density is satisfactorily recovered. Artificial by acetone, ethanol, and water, before observation.
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control of the ordered osteoblast alignment provides an
oriented microstructure for bone matrix formation, which 2.3. Mesenchymal stem cell culture
directs the requisite mechanical properties. Molecular Human mesenchymal stem cells from bone marrow
interactions between cells and materials play key roles (Takara Bio Inc., Shiga, Japan) were seeded at a
2
in the organization of the bone matrix. The key to concentration of 5000 cells/cm in MSC culture medium
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recovering lost bone function is the reconstruction of (Mesenchymal Stem Cell Growth Medium 2 [Takara]
the bone-mimetic anisotropic microstructure with fully mixed with SupplementMix MSC Growth Medium 2
differentiated osteoblasts. Additive manufacturing control [Takara], Penicillin Streptomycin [Gibco, Carlsbad, CA,
of a parallel groove structure is proposed here to control USA]). The cells were cultured at 37°C and 5.0% CO .
2
stem cell alignment as well as differentiation behaviors. The Cells were cultured for 1 day in short-term culture and
laser powder bed fusion (PBF-LB) method, a type of metal 4 weeks in long-term culture. In both short- and long-
additive manufacturing, enables elaborate 3D structural term cultures, the medium was replaced twice a week. All
control. PBF-LB is a powerful next-generation technology, operations were performed in a safety cabinet to prevent
and it fulfills the goal of using biomimetic materials bacterial contamination.
to create artificial organs. Indeed, our recent research 2.4. Immunofluorescent staining
developed an additive-manufactured vertebral fusion cage Mesenchymal stem cells were fixed by replacing the culture
device that can induce significantly superior bone fusion medium with 4% formalin/phosphate-buffered saline
without utilizing a large amount of autologous bone. 24,25 (PBS) and allowed to stand for 20 min. The cells were then
In this study, artificial control of the hMSCs washed twice with PBST (PBS 0.05% TritonX) for 10 min
differentiation lineage was achieved using a scaffold each time. The non-specific signals were blocked with 5%
surface structure, without any differentiation stimuli. normal goat serum (Invitrogen, Carlsbad, CA, USA) for 30
The additive-manufactured groove structure fulfilled min and then replaced with the primary antibody solution
the requirements for osteogenic differentiation of at 4°C overnight. The cells were washed and then incubated

Volume 10 Issue 1 (2024) 407 https://doi.org/10.18063/ijb.1425