The existing 3D printing methods exhibit certain fabrication-dependent limitations for printing curved constructs that are relevant for many tissues. Four-dimensional (4D) printing is an emerging technology that is expected to revolutionize the field of tissue engineering and regenerative medicine (TERM). 4D printing is based on 3D printing, featuring the introduction of time as the fourth dimension, in which there is a transition from a 3D printed scaffold to a new, distinct, and stable state, upon the application of one or more stimuli. Here, we present an overview of the current developments of the 4D printing technology for TERM, with a focus on approaches to achieve temporal changes of the shape of the printed constructs that would enable biofabrication of highly complex structures. To this aim, the printing methods, types of stimuli, shape-shifting mechanisms, and cell-incorporation strategies are critically reviewed. Furthermore, the challenges of this very recent biofabrication technology as well as the future research directions are discussed. Our findings show that the most common printing methods so far are stereolithography (SLA) and extrusion bioprinting, followed by fused deposition modelling, while the shape-shifting mechanisms used for TERM applications are shape-memory and differential swelling for 4D printing and 4D bioprinting, respectively. For shape-memory mechanism, there is a high prevalence of synthetic materials, such as polylactic acid (PLA), poly(glycerol dodecanoate) acrylate (PGDA), or polyurethanes. On the other hand, different acrylate combinations of alginate, hyaluronan, or gelatin have been used for differential swelling-based 4D transformations. TERM applications include bone, vascular, and cardiac tissues as the main target of the 4D (bio)printing technology. The field has great potential for further development by considering the combination of multiple stimuli, the use of a wider range of 4D techniques, and the implementation of computational-assisted strategies.

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The antibacterial biofunctionality of bone implants is essential for the prevention and treatment of implant-associated infections (IAI). In vitro co-culture models are utilized to assess this and study bacteria-host cell interactions at the implant interface, aiding our understanding of biomaterial and the immune response against IAI without impeding the peri-implant bone tissue regeneration. This paper reviews existing co-culture models together with their characteristics, results, and clinical relevance. A total of 36 studies were found involving in vitro co-culture models between bacteria and osteogenic or immune cells at the interface with orthopedic antibacterial biomaterials. Most studies (∼67%) involved co-culture models of osteogenic cells and bacteria (osteo-bac), while 33% were co-culture models of immune cells and bacterial cells (im-bac). All models involve direct co-culture of two different cell types. The cell seeding sequence (simultaneous, bacteria-first, and cell-first) was used to mimic clinically relevant conditions and showed the greatest effect on the outcome for both types of co-culture models. The im-bac models are considered more relevant for early peri-implant infections, whereas the osteo-bac models suit late infections. The limitations of the current models and future directions to develop more relevant co-culture models to address specific research questions are also discussed.@en

The currently available treatments for inner ear disorders often involve systemic drug administration, leading to suboptimal drug concentrations and side effects. Cochlear implants offer a potential solution by providing localized and sustained drug delivery to the cochlea. While the mechanical characterization of both the implants and their constituent material is crucial to ensure functional performance and structural integrity during implantation, this aspect has been mostly overlooked. This study proposes a novel methodology for the mechanical characterization of our recently developed cochlear implant design, namely, rectangular and cylindrical, fabricated using two-photon polymerization (2 PP) with a novel photosensitive resin (IP-Q™). We used in silico computational models and ex silico experiments to study the mechanics of our newly designed implants when subjected to torsion mimicking the foreseeable implantation procedure. Torsion testing on the actual-sized implants was not feasible due to their small size (0.6 × 0.6 × 2.4 mm³). Therefore, scaled-up rectangular cochlear implants (5 × 5 × 20 mm³, 10 × 10 × 40 mm³, and 20 × 20 × 80 mm³) were fabricated using stereolithography and subjected to torsion testing. Finite element analysis (FEA) accurately represented the linear behavior observed in the torsion experiments. We then used the validated Finite element analysis models to study the mechanical behavior of real-sized implants fabricated from the IP-Q resin. Mechanical characterization of both implant designs, with different inner porous structures (pore size: 20 μm and 60 μm) and a hollow version, revealed that the cylindrical implants exhibited approximately three times higher stiffness and mechanical strength as compared to the rectangular ones. The influence of the pore sizes on the mechanical behavior of these implant designs was found to be small. Based on these findings, the cylindrical design, regardless of the pore size, is recommended for further research and development efforts.@en

The Poisson's ratio and elastic modulus are two parameters determining the elastic behavior of biomaterials. While the effects of elastic modulus on the cell response is widely studied, very little is known regarding the effects of the Poisson's ratio. The micro-architecture of meta-biomaterials determines not only the Poisson's ratio but also several other parameters that also influence cell response, such as porosity, pore size, and effective elastic modulus. It is, therefore, very challenging to isolate the effects of the Poisson's ratio from those of other micro-architectural parameters. Here, we computationally design meta-biomaterials with controlled Poisson's ratios, ranging between -0.74 and +0.74, while maintaining consistent porosity, pore size, and effective elastic modulus. The 3D meta-biomaterials were additively manufactured at the micro-scale using two-photon polymerization (2PP), and were mechanically evaluated at the meso‑scale. The response of murine preosteoblasts to these meta-biomaterials was then studied using in vitro cell culture models. Meta-biomaterials with positive Poisson's ratios resulted in higher metabolic activity than those with negative values. The cells could attach and infiltrate all meta-biomaterials from the bottom to the top, fully covering the scaffolds after 17 days of culture. Interestingly, the meta-biomaterials exhibited different cell-induced deformations (e.g., shrinkage or local bending) as observed via scanning electron microscopy. The outcomes of osteogenic differentiation (i.e., Runx2 immunofluorescent staining) and matrix mineralization (i.e., Alizarin red staining) assays indicated the significant potential impact of these meta-biomaterials in the field of bone tissue engineering, paving the way for the development of advanced bone meta-implants. Statement of significance: We studied the influence of Poisson's ratio on bone cell response in meta-biomaterials. While elastic modulus effects are well-studied, the impact of Poisson's ratio, especially negative values found in architected biomaterials, remains largely unexplored. The complexity arises from intertwined micro-architectural parameters, such as porosity and elastic modulus, making it challenging to isolate the Poisson's ratio. To overcome this limitation, this study employed rational computational design to create meta-biomaterials with controlled Poisson's ratios, alongside consistent effective elastic modulus, porosity, and pore size. The study reveals that two-photon polymerized 3D meta-biomaterials with positive Poisson's ratios displayed higher metabolic activity, while all the developed meta-biomaterials supported osteogenic differentiation of preosteoblasts as well as matrix mineralization. The outcomes pave the way for the development of advanced 3D bone tissue models and meta-implants.

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This study proposes a new concept for an on-demand drug releasing device intended for integration into additively manufactured (i.e., 3D printed) orthopedic implants. The system comprises a surface with conduits connected to a subsurface reservoir used for storage and on-demand release of antimicrobial agents, covered with a cap that prevents the antibacterial agents from being released until alternating magnetic field (AMF) raises the temperature of the cap, thus, releasing the stored drug. To demonstrate this concept, Ti6Al4V specimens are directly 3D printed using selective laser melting and their surface, reservoirs, and drug releasing properties are characterized. A new synthetic antimicrobial peptide, SAAP-148, is thereafter tested for its cytotoxic, osteogenic, and immunomodulatory effects at concentrations relevant for its minimal bactericidal concentration (MBC) and is compared with its natural analogue, LL-37. The results showed that AMF successfully activated the release from the 3D printed loaded samples. Both peptides demonstrated to be non-cytotoxic within the MBC levels for macrophages and preosteoblasts and did not influence their osteoimmunomodulatory behavior. The findings of this study indicate that the proposed concept is technically feasible and has the potential to be used for the development of bone implants with on-demand delivery systems to fight IAI without systemic or continuous local release of antibiotics.

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Additively manufactured (AM) porous titanium implants may have an increased risk of implant-associated infection (IAI) due to their huge internal surfaces. However, the same surface, when biofunctionalized, can be used to prevent IAI. Here, we used a rat implant infection model to evaluate the biocompatibility and infection prevention performance of AM porous titanium against bioluminescent methicillin-resistant Staphylococcus aureus (MRSA). The specimens were biofunctionalized with Ag nanoparticles (NPs) using plasma electrolytic oxidation (PEO). Infection was initiated using either intramedullary injection in vivo or with in vitro inoculation of the implant prior to implantation. Nontreated (NT) implants were compared with PEO-treated implants with Ag NPs (PT-Ag), without Ag NPs (PT) and infection without an implant. After 7 days, the bacterial load and bone morphological changes were evaluated. When infection was initiated through in vivo injection, the presence of the implant did not enhance the infection, indicating that this technique may not assess the prevention but rather the treatment of IAIs. Following in vitro inoculation, the bacterial load on the implant and in the peri-implant bony tissue was reduced by over 90% for the PT-Ag implants compared to the PT and NT implants. All infected groups had enhanced osteomyelitis scores compared to the noninfected controls.@en

The development of biodegradable Fe-based bone implants has rapidly progressed in recent years. Most of the challenges encountered in developing such implants have been tackled individually or in combination using additive manufacturing technologies. Yet not all the challenges have been overcome. Herein, we present porous FeMn-akermanite composite scaffolds fabricated by extrusion-based 3D printing to address the unmet clinical needs associated with Fe-based biomaterials for bone regeneration, including low biodegradation rate, MRI-incompatibility, mechanical properties, and limited bioactivity. In this research, we developed inks containing Fe, 35 wt% Mn, and 20 or 30 vol% akermanite powder mixtures. 3D printing was optimized together with the debinding and sintering steps to obtain scaffolds with interconnected porosity of 69%. The Fe-matrix in the composites contained the γ-FeMn phase as well as nesosilicate phases. The former made the composites paramagnetic and, thus, MRI-friendly. The in vitro biodegradation rates of the composites with 20 and 30 vol% akermanite were respectively 0.24 and 0.27 mm/y, falling within the ideal range of biodegradation rates for bone substitution. The yield strengths of the porous composites stayed within the range of the values of the trabecular bone, despite in vitro biodegradation for 28 d. All the composite scaffolds favored the adhesion, proliferation, and osteogenic differentiation of preosteoblasts, as revealed by Runx2 assay. Moreover, osteopontin was detected in the extracellular matrix of cells on the scaffolds. Altogether, these results demonstrate the remarkable potential of these composites in fulfilling the requirements of porous biodegradable bone substitutes, motivating future in vivo research. Statement of significance: We developed FeMn-akermanite composite scaffolds by taking advantage of the multi-material capacity of extrusion-based 3D printing. Our results demonstrated that the FeMn-akermanite scaffolds showed an exceptional performance in fulfilling all the requirements for bone substitution in vitro, i.e., a sufficient biodegradation rate, having mechanical properties in the range of trabecular bone even after 4 weeks biodegradation, paramagnetic, cytocompatible and most importantly osteogenic. Our results encourage further research on Fe-based bone implants in in vivo.

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Living organisms use functional gradients (FGs) to interface hard and soft materials (e.g., bone and tendon), a strategy with engineering potential. Past attempts involving hard (or soft) phase ratio variation have led to mechanical property inaccuracies because of microscale-material macroscale-property nonlinearity. This study examines 3D-printed voxels from either hard or soft phase to decode this relationship. Combining micro/macroscale experiments and finite element simulations, a power law model emerges, linking voxel arrangement to composite properties. This model guides the creation of voxel-level FG structures, resulting in two biomimetic constructs mimicking specific bone-soft tissue interfaces with superior mechanical properties. Additionally, the model studies the FG influence on murine preosteoblast and human bone marrow-derived mesenchymal stromal cell (hBMSC) morphology and protein expression, driving rational design of soft-hard interfaces in biomedical applications.

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Durable interfacing of hard and soft materials is a major design challenge caused by the ensuing stress concentrations. In nature, soft-hard interfaces exhibit remarkable mechanical performance, with failures rarely happening at the interface. Here, we mimic the strategies observed in nature to design efficient soft-hard interfaces. We base our geometrical designs on triply periodic minimal surfaces (i.e., Octo, Diamond, and Gyroid), collagen-like triple helices, and randomly distributed particles. A combination of computational simulations and experimental techniques, including uniaxial tensile and quad-lap shear tests, are used to characterize the mechanical performance of the interfaces. Our analyses suggest that smooth interdigitated connections, compliant gradient transitions, and either decreasing or constraining strain concentrations lead to simultaneously strong and tough interfaces. We generate additional interfaces where the abovementioned toughening mechanisms work synergistically to create soft-hard interfaces with strengths approaching the upper achievable limit and enhancing toughness values by 50%, as compared to the control group.

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Individual cells and multicellular systems respond to cell-scale curvatures in their environments, guiding migration, orientation, and tissue formation. However, it remains largely unclear how cells collectively explore and pattern complex landscapes with curvature gradients across the Euclidean and non-Euclidean spectra. Here, we show that mathematically designed substrates with controlled curvature variations induce multicellular spatiotemporal organization of preosteoblasts. We quantify curvature-induced patterning and find that cells generally prefer regions with at least one negative principal curvature. However, we also show that the developing tissue can eventually cover unfavorably curved territories, can bridge large portions of the substrates, and is often characterized by collectively aligned stress fibers. We demonstrate that this is partly regulated by cellular contractility and extracellular matrix development, underscoring the mechanical nature of curvature guidance. Our findings offer a geometric perspective on cell-environment interactions that could be harnessed in tissue engineering and regenerative medicine applications.

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Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3 g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.@en

Hearing loss is a highly prevalent multifactorial disorder affecting 20% of the global population. Current treatments using the systemic administration of drugs are therapeutically ineffective due to the anatomy of the cochlea and the existing blood–labyrinth barrier. Local drug delivery systems can ensure therapeutic drug concentrations locally while preventing adverse effects caused by high dosages of systemically administered drugs. Here, we aimed to design, fabricate, and characterize a local drug delivery system for the human cochlea. The design was relevant to the size of the human ear, included two different shapes, and incorporated two different microporous structures acting as reservoirs for drug loading and release. The four cochlear implant designs were printed using the two-photon polymerization (2PP) technique and the IP-Q photoresist. The optimized 2PP process enabled the fabrication of the cochlear implants with great reproducibility and shape fidelity. Rectangular and cylindrical implants featuring cylindrical and tapered tips, respectively, were successfully printed. Their outer dimensions were 0.6 × 0.6 × 2.4 mm3 (L × W × H). They incorporated internal porous networks that were printed with high accuracy, yielding pore sizes of 17.88 ± 0.95 μm and 58.15 ± 1.62 μm for the designed values of 20 μm and 60 μm, respectively. The average surface roughness was 1.67 ± 0.24 μm, and the water contact angle was 72.3 ± 3.0°. A high degree of polymerization (~90%) of the IP-Q was identified after printing, and the printed material was cytocompatible with murine macrophages. The cochlear implants designed and 3D printed in this study, featuring relevant sizes for the human ear and tunable internal microporosity, represent a novel approach for personalized treatment of hearing loss through local drug delivery.@en

Modulation of the immune response following the implantation of biomaterials can have beneficial effects on bone regeneration. This involves complex interactions between the inflammatory and osteogenic cells. Therefore, the study of cell-cell interactions using direct co-culture models integrated with biomaterials is of great interest. This research aimed to study the viability, morphology, and osteogenic activity of preosteoblasts (OBs) co-cultured with pro-inflammatory macrophages (M1s) on the 3D printed (non)patterned surfaces. OBs and M1s remained alive and proliferated actively for 14 days in the mixture of Dulbecco's Modified Eagle's Medium (DMEM) and alpha Minimum Essential Medium (α-MEM) (1:1), regardless of the cell ratio in the co-cultures. The spatial organization of the two types of cells changed with the time of culture from an initially uniform cell distribution to the formation of a thick layer of OBs covered by clusters of M1s. On day 7, the expression of PGE2 and TNF-α were upregulated in the co-culture relative to the mono-culture of OBs and M1s. The inflammation decreased differentiation and matrix mineralization of OBs after 28 days of culture. Interestingly, the incorporation of 3D printed submicron pillars into the direct co-culture model enhanced the differentiation of preosteoblasts, as shown by relatively higher RUNX2 expression, thereby revealing the osteoimmunomodulatory potential of such surface patterns.

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3D bioprinting is usually implemented on flat surfaces, posing serious limitations in the fabrication of multilayered curved constructs. 4D bioprinting, combining 3D bioprinting with time-dependent stimuli-induced transformation, enables the fabrication of shape-changing constructs. Here, a 4D biofabrication method is reported for cartilage engineering based on the differential swelling of a smart multi-material system made from two hydrogel-based materials: hyaluronan and alginate. Two ink formulations are used: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Both inks have similar elastic, shear-thinning, and printability behavior. The inks are 3D printed into a bilayered scaffold before triggering the shape-change by using liquid immersion as stimulus. In time (4D), the differential swelling between the two zones leads to the scaffold's self-bending. Different designs are made to tune the radius of curvature and shape. A bioprinted formulation of AHAT and human bone marrow cells demonstrates high cell viability. After 28 days in chondrogenic medium, the curvature is clearly present while cartilage-like matrix production is visible on histology. A proof-of-concept of the recently emerged technology of 4D bioprinting with a specific application for the design of curved structures potentially mimicking the curvature and multilayer cellular nature of native cartilage is demonstrated.

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Implant-associated infections are highly challenging to treat, particularly with the emergence of multidrug-resistant microbials. Effective preventive action is desired to be at the implant site. Surface biofunctionalization of implants through Ag-doping has demonstrated potent antibacterial results. However, it may adversely affect bone regeneration at high doses. Benefiting from the potential synergistic effects, combining Ag with other antibacterial agents can substantially decrease the required Ag concentration. To date, no study has been performed on immobilizing both Ag and Fe nanoparticles (NPs) on the surface of additively manufactured porous titanium. We additively manufactured porous titanium and biofunctionalized its surface with plasma electrolytic oxidation using a Ca/P-based electrolyte containing Fe NPs, Ag NPs, and the combinations. The specimen’s surface morphology featured porous TiO2 bearing Ag and Fe NPs. During immersion, Ag and Fe ions were released for up to 28 days. Antibacterial assays against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa showed that the specimens containing Ag NPs and Ag/Fe NPs exhibit bactericidal activity. The Ag and Fe NPs worked synergistically, even when Ag was reduced by up to three times. The biofunctionalized scaffold reduced Ag and Fe NPs, improving preosteoblasts proliferation and Ca-sensing receptor activation. In conclusion, surface biofunctionalization of porous titanium with Ag and Fe NPs is a promising strategy to prevent implant-associated infections and allow bone regeneration and, therefore, should be developed for clinical application.@en

We designed and fabricated a simple setup for the controlled crumpling of nanopatterned, surface-porous flat metallic sheets for the fabrication of volume-porous biomaterials and showed that crumpling can be considered as an efficient alternative to origami-inspired folding. Before crumpling, laser cutting was used to introduce pores to the sheets. We then fabricated titanium (Ti) nanopatterns through reactive ion etching on the polished Ti sheets. Thereafter, nanopatterned porous Ti sheets were crumpled at two deformation velocities (i.e., 2 and 100 mm/min). The compression tests of the scaffolds indicated that the elastic modulus of the specimens vary in the range of 11.8–13.9 MPa. Micro-computed tomography scans and computational simulations of crumpled scaffolds were performed to study the morphological properties of the resulting meta-biomaterials. The porosity and pore size of the scaffolds remained within the range of those reported for trabecular bone. Finally, the in vitro cell preosteoblasts culture demonstrated the cytocompatibility of the nanopatterned scaffolds. Moreover, the aspect ratio of the cells residing on the nanopatterned surfaces was found to be significantly higher than those cultured on the control scaffolds, indicating that the nanopatterned surface may have a higher potential for inducing the osteogenic differentiation of the preosteoblasts.

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Physical patterns represent potential surface cues for promoting osteogenic differentiation of stem cells and improving osseointegration of orthopedic implants. Understanding the early cell–surface interactions and their effects on late cellular functions is essential for a rational design of such topographies, yet still elusive. In this work, fluidic force microscopy (FluidFM) and atomic force microscopy (AFM) combined with optical and electron microscopy are used to quantitatively investigate the interaction of preosteoblasts with 3D-printed patterns after 4 and 24 h of culture. The patterns consist of pillars with the same diameter (200 nm) and interspace (700 nm) but distinct heights (500 and 1000 nm) and osteogenic properties. FluidFM reveals a higher cell adhesion strength after 24 h of culture on the taller pillars (32 ± 7 kPa versus 21.5 ± 12.5 kPa). This is associated with attachment of cells partly on the sidewalls of these pillars, thus requiring larger normal forces for detachment. Furthermore, the higher resistance to shear forces observed for these cells indicates an enhanced anchorage and can be related to the persistence and stability of lamellipodia. The study explains the differential cell adhesion behavior induced by different pillar heights, enabling advancements in the rational design of osteogenic patterns.

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Folding nanopatterned flat sheets into complex 3D structures enables the fabrication of meta-biomaterials that combine a rationally designed 3D architecture with nanoscale surface features. Self-folding is an attractive approach for realizing such materials. However, self-folded lattices are generally too compliant as there is an inherent competition between ease-of-folding requirements and final load-bearing characteristics. Inspired by sheet metal forming, an alternative route is proposed for the fabrication of origamilattices. This ‘automated-folding’ approach allows for the introduction of sharp folds into thick metal sheets, thereby enhancing their stiffness. The first time realization of automatically folded origami lattices with bone-mimicking mechanical properties is demonstrated. The proposed approach is highly scalable given that the unit cells making up the meta-biomaterial can be arbitrarily large in number and small in dimensions. To demonstrate the scalability and versatility of the proposed approach, it is fabricated origamilattices with > 100 unit cells, lattices with unit cells as small as 1.25 mm, and auxetic lattices. The nanopatterned the surface of the sheets prior to folding. Protected by a thin coating layer, these nanoscale features remained intact during the folding process. It is found that the nanopatterned folded specimens exhibits significantly increased mineralization as compared to their non-patterned counterparts.

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Additively manufacturing of porous iron offers a unique opportunity to increase its biodegradation rate by taking advantage of arbitrarily complex porous structures. Nevertheless, achieving the required biodegradation profile remains challenging due to the natural passivation of iron that decrease the biodegradation rate. Moreover, the biocompatibility of iron is reported to be limited. Here, we address both challenges by applying poly(2-ethyl-2-oxazoline) coating to extrusion-based 3D printed porous iron. We characterized the specimens by performing in vitro biodegradation, electrochemical measurements, time-dependent mechanical tests, and in vitro cytocompatibility assays. The coated porous iron exhibited a biodegradation rate that was 2.6× higher than that of non-coated counterpart and maintained the bone-mimicking mechanical properties throughout biodegradation. Despite the formation of dense biodegradation products, the coating ensured a relatively stable biodegradation (i.e., 17% reduction in the degradation rate between days 14 and 28) as compared to that of non-coated specimens (i.e., 43% drop). Furthermore, the coating could be identified even after biodegradation, demonstrating the longevity of the coating. Finally, the coated specimens significantly increased the viability and supported the attachment and growth of preosteoblasts. Our results demonstrate the great potential of poly(2-ethyl-2-oxazoline) coating for addressing the multiple challenges associated with the clinical adoption of porous iron.

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Developing high-throughput nanopatterning techniques that also allow for precise control over the dimensions of the fabricated features is essential for the study of cell-nanopattern interactions. Here, we developed a process that fulfills both of these criteria. Firstly, we used electron-beam lithography (EBL) to fabricate precisely controlled arrays of submicron pillars with varying values of interspacing on a large area of fused silica. Two types of etching procedures with two different systems were developed to etch the fused silica and create the final desired height. We then studied the interactions of preosteoblasts (MC3T3-E1) with these pillars. Varying interspacing was observed to significantly affect the morphological characteristics of the cell, the organization of actin fibers, and the formation of focal adhesions. The expression of osteopontin (OPN) significantly increased on the patterns, indicating the potential of the pillars for inducing osteogenic differentiation. The EBL pillars were thereafter used as master molds in two subsequent processing steps, namely soft lithography and thermal nanoimprint lithography for high-fidelity replication of the pillars on the substrates of interest. The molding parameters were optimized to maximize the fidelity of the generated patterns and minimize the wear and tear of the master mold. Comparing the replicated feature with those present on the original mold confirmed that the geometry and dimensions of the replicated pillars closely resemble those of the original ones. The method proposed in this study, therefore, enables the precise fabrication of submicron- and nanopatterns on a wide variety of materials that are relevant for systematic cell studies. Statement of significance: Submicron pillars with specific dimensions on the bone implants have been proven to be effective in controlling cell behaviors. Nowadays, numerous methods have been proposed to produce bio-instructive submicron-topographies. However, most of these techniques are suffering from being low-throughput, low-precision, and expensive. Here, we developed a high-throughput nanopatterning technique that allows for control over the dimensions of the features for the study of cell-nanotopography interactions. Assessing the adaptation of preosteoblast cells showed the potential of the pillars for inducing osteogenic differentiation. Afterward, the pillars were used for high-fidelity replication of the bio-instructive features on the substrates of interest. The results show the advantages of nanoimprint lithography as a unique technique for the patterning of large areas of bio-instructive surfaces.

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