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International Journal of Bioprinting Stiffness of scaffold-mediated immune response

inflammatory response through the secretion of cytokines of intricate structures at a smaller scale. Extrusion-
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such as interleukin-6 (IL-6), inducible nitric oxide synthase based bioprinting, which is one of the most commonly
(iNOS), and tumor necrosis factor alpha (TNF-α). They used bioprinting technologies, permits the printing of
6,7
also recruit lymphocytes involved in adaptive immune scaffolds with complex structures by depositing materials
responses. As wound healing progresses, M2 macrophages layer by layer. By utilizing 3D printing technology,
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become the dominant immune cells. These M2 macrophages researchers can overcome the limitations of traditional
8
express anti-inflammatory factors like interleukin-10 (IL- approaches and create biomimetic scaffolds that closely
10), arginase-1 (Arg1), and transforming growth factor beta mimic the native tissue environment. This not only
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(TGF-β), which promote tissue regeneration. However, provides more accurate and relevant models for studying
9
if inflammation persists, macrophages can fuse to form the interplay between biomaterial stiffness and immune
foreign body macrophages. This fusion induces adverse response but also ensures a high degree of reproducibility
immune responses, such as the foreign body reaction (FBR), in the complex structures of tissues and organs. Alginate
which leads to the formation of a fibrotic capsule around and gelatin are natural polymers commonly used in
the scaffold. 10,11 This exacerbates tissue damage and may extrusion-based bioprinting. 25,26 By adjusting the ratio
even result in implant failure. Therefore, achieving a balance of gelatin to alginate, the physical properties of alginate–
between inflammatory activation and suppression is crucial gelatin composite hydrogels can be modulated. Previous
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for developing effective immunomodulatory biomaterials, research has demonstrated that alginate–gelatin composite
which are vital for advancing tissue engineering toward hydrogels exhibit excellent biocompatibility and that
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clinical applications.
3D-bioprinted scaffolds based on these hydrogels can
Various strategies have been employed to regulate regulate the differentiation fate of mesenchymal stem
the immune response to biomaterial scaffolds, including cells (MSCs). However, the immune response elicited by
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modifying their physical or chemical properties and these 3D-printed scaffolds has been largely unexplored,
incorporating immunomodulatory factors. 12,13 Among hindering their clinical translation. Consequently, it is
these strategies, material stiffness has received significant crucial to investigate the immune responses induced by
attention. 14,15 Macrophages respond to the mechanical alginate–gelatin composite hydrogel scaffolds.
properties of their microenvironment. Some studies have
suggested that increased matrix stiffness leads to increased In this study, we fabricated three types of 3D-bioprinted
expression of inflammatory factors such as TNF-α and scaffolds with varying stiffness by adjusting the ratio of
interleukin-1β (IL-1β) by macrophages, both with and alginate to gelatin. We investigated the physical properties
without LPS stimulation, in vitro. 15-17 However, other of these hydrogel scaffolds and assessed the biocompatibility
studies have reported an anti-inflammatory phenotype and polarization response of RAW264.7 macrophages
in softer hydrogels and a pro-inflammatory phenotype encapsulated within hydrogels of different stiffness.
in stiffer hydrogels. 18,19 These findings indicate that the Furthermore, we implanted the scaffolds subcutaneously in
interaction between biomaterials and macrophages is mice to explore the immune responses in vivo. Additionally,
complex, and our understanding of the immune responses we conducted a preliminary RNA sequencing (RNA-seq)
mediated by biomaterials in vivo remains limited. study to elucidate potential mechanisms, thereby providing
guidance for the application of 3D-bioprinted scaffolds.
The current research on the interplay between
biomaterial stiffness and immune response primarily 2. Methods and materials
focuses on the biomaterials themselves. However,
unstructured biomaterials lack sufficient biomimetic 2.1. Bioink preparation
properties. Three-dimensional (3D) printing technology The bioinks S1, S2, and S3 were prepared according to the
offers a solution by enabling the spatial integration of a wide compositions listed in Table 1. Gelatin and alginate were
range of biological, physical, and biochemical signals to dissolved in ddH2O at 70°C until thoroughly mixed. Then,
modulate cells. According to the criteria set by the American the bioinks were pasteurized and stored at 4°C.
Society for Testing and Materials (ASTM), 3D bioprinting
technologies can be classified into three categories: jetting- 2.2. Cell culture
based, extrusion-based, and vat polymerization-based The mouse-derived macrophage cell line RAW264.7 cells
bioprinting. Inkjet-based bioprinting has been shown to (C7505, Beyotime, China) were cultured in high-glucose
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have higher cell viability, as it enables the precise deposition Dulbecco’s Modified Eagle Medium (DMEM, Biological
of cells and biomaterials in a controlled manner. 20,21 Industries, Israel) supplemented with 10% fetal bovine
Reduced photopolymerization bioprinting, on the other serum (FBS; Gibco, USA) at 37°C and 5% CO . To avoid
2
hand, offers higher resolution, allowing for the fabrication polarization of RAW264.7, the medium was renewed

Volume 10 Issue 4 (2024) 338 doi: 10.36922/ijb.2874