Biodegradable zinc (Zn)-based materials offer promising applications in orthopedics due to their excellent mechanical properties, osteogenic, and antibacterial bioactivities. However, a critical challenge arises from the mismatch between the slow degradation rate of bulk Zn implants (a decade or more) and the relatively short timeframe of bone repair (3–6 months). 3D printing offers a solution by creating porous scaffolds with reduced material consumption, but this introduces a new problem: accelerated degradation and the potential for zinc overdose toxicity due to increased surface area. This study aims to overcome this dilemma through a multiscale architectural design of Zn-based scaffolds, incorporating osteoimmunomodulation to precisely control degradation and promote bone regeneration. The immune system plays a crucial role in bone regeneration. Biomaterials trigger an inflammatory response, and materials with osteoimmunomodulatory properties can modulate immune cell behavior for favorable tissue responses. Macrophages, in particular, are key players in bone regeneration, and their polarization (M1 pro-inflammatory vs. M2 anti-inflammatory) is critical. The design of porous scaffolds needs to consider this interaction at multiple scales: microscale (ion release and cellular uptake), mesoscale (surface topography and cell adhesion), and macroscale (pore geometry and overall scaffold degradation). This study leverages these principles to develop a novel Zn-based scaffold design with optimized osteoimmunomodulatory properties for improved bone regeneration.

Numerous studies have explored Zn-based biomaterials for orthopedic applications, including screw and plate systems, intramedullary needles, bone grafts, and guided bone regeneration membranes. Clinical trials have also been conducted using zinc alloy implants. However, the slow degradation of bulk Zn implants poses a significant challenge. Recent advancements in 3D printing technology have enabled the fabrication of porous Zn-based scaffolds, aiming to reduce material consumption and improve degradation kinetics. While promising, the increased surface area of porous scaffolds can lead to accelerated degradation and zinc toxicity. The role of immune cells, especially macrophages, in bone regeneration is well-established, and the concept of osteoimmunomodulation, which aims to modulate immune cell behavior for favorable tissue responses, is gaining traction. Previous studies have demonstrated the influence of surface topography and pore geometry on cell behavior and material degradation. However, a systematic, multiscale approach to designing Zn-based scaffolds that considers both degradation and osteoimmunomodulation is lacking.

This study employed a multiscale approach to design and fabricate Zn-based porous scaffolds. **Composition Design:** The researchers investigated Zn-Li alloys, varying the Li content to find the optimal balance between mechanical strength and plasticity, and to control the release of Zn and Li ions and their effects on macrophage polarization. The selected alloy (Zn-0.8Li) underwent atomization to create powder for 3D printing. **3D Printing and Surface Modification:** Laser Powder Bed Fusion (L-PBF) was used to fabricate porous scaffolds with two distinct pore geometries: a body-centered cubic (BCC) structure and a gyroid (G) structure. Ultrasonic treatment, acid etching, and electrochemical polishing were employed to modify the scaffold surface, creating different surface patterns and roughness. Atomic force microscopy (AFM) characterized surface roughness. **Characterization:** The microstructure of the Zn-0.8Li alloy was examined using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Electrochemical behavior was assessed using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization (PDP) curves, and scanning vibrating electrode technique (SVET). The degradation behavior was investigated by dynamic immersion testing in simulated body fluid (SBF) and the release of Zn and Li ions were quantified using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). X-ray photoelectron spectroscopy (XPS) was used to analyze corrosion products. **In Vitro Studies:** The biocompatibility of the scaffolds was evaluated using live/dead assays, SEM, and F-actin staining of RAW264.7 (macrophages) and MC3T3-E1 (pre-osteoblasts) cells. The effect of scaffold extracts on macrophage polarization was assessed using immunofluorescence staining (iNOS, CD206) and qRT-PCR for inflammatory cytokines (IL-4, IL-10, Arg1, TNF-α, iNOS, IL-1β). Transcriptomic analysis (RNA-seq) was performed to identify differentially expressed genes and associated pathways. The effect of macrophage-conditioned medium (CM) on osteogenic differentiation of MC3T3-E1 cells was evaluated using ALP staining, ARS staining, and qRT-PCR for osteogenic markers (ALP, OPG, OPN, Col1a1). **In Vivo Studies:** The in vivo degradation and bone regeneration were studied by implanting the scaffolds into rat femoral condyles. X-ray fluorescence imaging (XRF), SEM, micro-CT, and histological analysis (methylene blue acid fuchsin staining, immunohistochemistry for iNOS and CD163, ALP and OCN staining) were performed at 3 days, 1 month, and 3 months post-implantation. Second-harmonic generation (SHG) microscopy was used to visualize collagen fiber orientation.

The study yielded several key findings: **Optimal Alloy Composition:** The Zn-0.8Li alloy exhibited the optimal balance between mechanical strength and plasticity and effectively modulated macrophage polarization. The co-release of Zn and Li ions from Zn-0.8Li alloy promotes the polarization of non-activated macrophages (MO) to macrophages with a pro-regenerative phenotype (M2) and stimulates the expression of immunomodulatory cytokines most efficiently. **Surface Patterning:** Electrochemical polishing created a wavy-like surface with nanoscale roughness (Ra=114 nm), significantly improving cell adhesion and spreading, particularly in RAW264.7 macrophages. **Scaffold Geometry:** The gyroid (G) scaffold exhibited superior mechanical properties compared to the BCC scaffold under compression and demonstrated more uniform degradation with longer maintenance of mechanical integrity. The G scaffold demonstrated a more uniform degradation profile and superior maintenance of structural integrity compared to the BCC scaffold. **In Vitro Osteoimmunomodulation:** Extracts from the G scaffold promoted M2 macrophage polarization (increased CD206, decreased iNOS), upregulated anti-inflammatory cytokines (IL-4, IL-10, Arg1), and downregulated pro-inflammatory cytokines (TNF-α, iNOS, IL-1β). Transcriptomic analysis revealed activation of the JAK/STAT pathway and downregulation of MAPK pathways in macrophages treated with G scaffold extracts. Conditioned medium from these macrophages significantly enhanced osteogenic differentiation of MC3T3-E1 cells. **In Vivo Bone Regeneration:** In vivo, the G scaffold showed more uniform degradation and significantly greater bone ingrowth and bone volume fraction (BV/TV) compared to the BCC scaffold at 3 months. The G scaffold also promoted a faster shift from M1 to M2 macrophage polarization at the implant site and exhibited greater expression of osteogenic markers (ALP, OCN). Collagen fibers were well-aligned along the curved struts of the G scaffold, while in BCC scaffolds, collagen distribution was more point-like.

The results demonstrate the successful implementation of a multiscale design strategy for biodegradable Zn-based scaffolds to enhance bone regeneration. The integration of materials science, immunology, and 3D printing technologies allowed for the creation of a scaffold that addresses the limitations of previous Zn-based implants. The optimal composition of the Zn-0.8Li alloy and the unique surface patterning and pore geometry of the G scaffold are key contributors to the improved performance. The observed osteoimmunomodulatory effects, leading to increased bone formation, are a significant advancement. The findings highlight the importance of considering the interaction between biomaterials and the immune system at multiple scales. Future studies could explore other alloying elements or surface modifications to further optimize the scaffold's properties. The potential clinical translation of these scaffolds warrants further investigation in larger animal models and clinical trials.

This study demonstrates that a multiscale design approach, combining optimized alloy composition, surface modification, and scaffold geometry, can lead to significantly improved bone regeneration using 3D-printed biodegradable Zn-based scaffolds. The gyroid (G) scaffold, in particular, shows great promise due to its enhanced mechanical properties, uniform degradation, and ability to promote osteoimmunomodulation. Future studies should focus on clinical translation through larger animal models and ultimately human clinical trials. Investigation of other alloying elements to further enhance bioactivity or mechanical properties and exploration of different pore architectures could further optimize scaffold design.

The study used a rat femoral condyle defect model, which may not fully represent the complexity of bone defects in humans. The sample size in the in vivo study was relatively small. Long-term in vivo studies are needed to fully assess the degradation kinetics and long-term biocompatibility of the scaffolds. The transcriptomic analysis focused on a limited number of genes and pathways and a more comprehensive analysis could provide further insights into the mechanisms of action. Future studies should be performed on larger animals such as sheep or dogs to fully verify the results.