Yttria-stabilized tetragonal zirconia polycrystals (Y-TZPs) fabricated through subtractive manufacturing (SM) have become the material of choice for dental restorations, including crowns, frameworks, implant fixtures, and abutments [1], [2], [3], [4]. Their popularity stems from their superior mechanical properties [5], esthetic compatibility [5], [6], biocompatibility with soft tissues [7], low inflammation response [8], and reliable osseointegration capabilities [9]. However, SM technology, which involves milling zirconia from pre-sintered blocks, presents significant limitations, such as excessive material waste (up to 80 wt%) [10], micro-crack formation [11], and restricted geometric complexity due to milling tool size and axis constraints [12], [13]. These challenges hinder the fabrication of advanced prostheses requiring intricate designs.

Additive manufacturing (AM), also known as three-dimensional (3D) printing, has been developing rapidly as a promising alternative to SM, offering advantages such as reduced material waste and the ability to create complex geometries [12]. According to ISO/ASTM 52900:2021 standards, AM technologies are classified into seven categories, with vat photopolymerization (VP) and material jetting (MJ) being particularly suitable for dental zirconia prostheses [14], [15]. VP, including stereolithography (SLA) and digital light processing (DLP), uses ultraviolet (UV) light to selectively polymerize photopolymerizable zirconia slurries [15], [16]. SLA employs a laser beam for point-wise curing, while DLP polymerizes entire layers simultaneously via a digital micromirror device (DMD), significantly enhancing printing speed [17], [18]. MJ, often referred to as “Nanoparticle jetting”, deposits zirconia nanoparticle-laden droplets alongside soluble support materials, forming a green body that is later post-processed via a demineralized water bath [19], [20], [21]. Studies report that MJ achieves superior dimensional accuracy and interfacial bond strength compared to traditional methods [21], [22], [23], [24], [25].

Mechanically, AM-fabricated zirconia meets or exceeds ISO 6872 standards for multi-unit restorations (flexural strength＞800 MPa), with reported values surpassing 1500 MPa—outperforming SM counterparts (∼980 MPa) [26], [27]. Despite these advancements, biological evaluations remain limited, particularly regarding soft-tissue interactions. Existing research primarily focuses on osteoblast responses to evaluate the osseointegration potential of zirconia implants produced by specific AM technologies [19], [28], [29], [30], with limited attention to gingival cells [31], [32]. This gap is critical, as soft-tissue integration is essential for implants and abutments to prevent bacterial infiltration, mitigate peri-implantitis, and ensure long-term clinical success [33], [34], [35]. Additionally, strong soft-tissue adhesion is equally vital for crowns and frameworks that directly contact gingival tissue.

Gingival fibroblasts, the primary cells in gingival connective tissue, play a crucial role in mucosal seal formation via adhesion, proliferation, migration, and extracellular matrix (ECM) synthesis [33]. Surface topography—a key factor influencing cellular behaviors via "contact guidance" (directional cell alignment in response to structural cues)—varies significantly across AM technologies and build orientations [19], [36], [37], [38], [39]. For example, Zhang et al. [19] observed that MJ-fabricated implants built at 0° orientation exhibited a distinct parallel lamellar structure along the printing layers. This directional surface structure aligned cellular morphology and enhanced osteoblast proliferation and matrix mineralization, suggesting similar effects on gingival fibroblasts.

Therefore, in this study, the surface properties of different zirconia specimens were characterized, and the responses of human gingival fibroblasts (HGFs), including cytotoxicity, viability, morphology, adhesion, proliferation, and migration, were evaluated. Additionally, RNA sequencing (RNA-Seq) and transcriptome bioinformatics analysis were conducted to explore gene expression changes in cells exhibiting optimal behaviors, providing deeper insights into the effects of AM-fabricated zirconia on gingival tissue. The aim of the present study was to evaluate the responses of HGFs to zirconia fabricated using 3 AM technologies (SLA, DLP and MJ) at horizontal (0°) and vertical (90°) orientations, compared to SM controls. The first null hypothesis tested was that there would be no significant differences in HGF responses to zirconia fabricated using different AM technologies or build orientations. The second null hypothesis was that zirconia fabricated via AM and SM would elicit comparable HGF responses.