Scaffold Geometry Research Articles

Application and progress of 3D printed biomaterials in osteoporosis

Osteoporosis results from a disruption in skeletal homeostasis caused by an imbalance between bone resorption and bone formation. Conventional treatments, such as pharmaceutical drugs and hormone replacement therapy, often yield suboptimal results and are frequently associated with side effects. Recently, biomaterial-based approaches have gained attention as promising alternatives for managing osteoporosis. This review summarizes the current advancements in 3D-printed biomaterials designed for osteoporosis treatment. The benefits of biomaterial-based approaches compared to traditional systemic drug therapies are discussed. These 3D-printed materials can be broadly categorized based on their functionalities, including promoting osteogenesis, reducing inflammation, exhibiting antioxidant properties, and inhibiting osteoclast activity. 3D printing has the advantages of speed, precision, personalization, etc. It is able to satisfy the requirements of irregular geometry, differentiated composition, and multilayered structure of articular osteochondral scaffolds with boundary layer structure. The limitations of existing biomaterials are critically analyzed and future directions for biomaterial-based therapies are considered.

Scaffolds fabricated via two-photon polymerisation for regulating cell morphology and differentiation

ABSTRACT Three-dimensional (3D) cell culture scaffolds play a key role in guiding cell fate. The fine-tunability of these scaffolds is essential for accurately replicating in vivo conditions and revealing cellular behaviours. In this study, two-photon polymerisation (TPP) technology was employed to create 3D scaffolds with woodpile and honeycomb architectures. The influence of scaffold geometry and pore dimensions on the proliferation, morphology, orientation, and differentiation of human mesenchymal stem cells (hMSCs) was investigated through multi-staining fluorescence and 3D confocal imaging technique. It was revealed that hMSCs cultured within these 3D scaffolds demonstrated higher density up to 15.04 ± 1.46 cells/100 × 100 μm2 after a 6-day culture compared to their 2D counterparts (7.47 ± 1.42 cells/100 × 100 μm2). Furthermore, hMSCs grown on the woodpile scaffolds displayed elongated morphology, with approximately 50% aligning along the columns. In contrast, hMSCs cultivated on honeycomb structures exhibited triangular and crescent cellular shapes, with a random orientation. Notably, compared to the control group, the cells on the scaffolds exhibited smaller nucleus areas, lower circularity, and aspect ratios, but these values increased as pore size increased. Furthermore, ALP staining showed a greater tendency for osteogenic differentiation with larger pore sizes. These TPP-fabricated 3D scaffolds hold immense promise for advancing understanding of cellular behaviour.

Predicting inflammatory response of biomimetic nanofibre scaffolds for tissue regeneration using machine learning and graph theory.

Tissue regeneration after a wound occurs through three main overlapping and interrelated stages namely inflammatory, proliferative, and remodelling phases, respectively. The inflammatory phase is key for successful tissue reconstruction and triggers the proliferative phase. The macrophages in the non-healing wounds remain in the inflammatory loop, but their phenotypes can be changed via interactions with nanofibre-based scaffolds mimicking the organisation of the native structural support of healthy tissues. However, the organisation of extracellular matrix (ECM) is highly complex, combining order and disorder, which makes it difficult to replicate. The possibility of predicting the desirable biomimetic geometry and chemistry of these nanofibre scaffolds would streamline the scaffold design process. Fifteen families of nanofibre scaffolds, electrospun from combinations of polyesters (polylactide, polyhydroxybutyrate), polysaccharides (polysucrose, carrageenan, cellulose), and polyester ether (polydioxanone) were investigated and analysed using machine learning (ML). The Random Forest model had the best performance (92.8%) in predicting inflammatory responses of macrophages on the nanoscaffolds using tumour necrosis factor-alpha as the output. CellProfiler proved to be an effective tool to process scanning electron microscopy (SEM) images of the macrophages on the scaffolds, successfully extracting various features and measurements related to cell phenotypes M0, M1, and M2. Deep learning modelling indicated that convolutional neural network models have the potential to be applied to SEM images to classify macrophage cells according to their phenotypes. The complex organisation of the nanofibre scaffolds can be analysed using graph theory (GT), revealing the underlying connectivity patterns of the nanofibres. Analysis of GT descriptors showed that the electrospun membranes closely mimic the connectivity patterns of the ECM. We conclude that ML-facilitated, GT-quantified engineering of cellular scaffolds has the potential to predict cell interactions, streamlining the pipeline for tissue engineering.

A Trithia‐Bridged N‐Heterotriangulene: The Hitherto Missing Electron Donor

AbstractThe very first representative of trithia‐bridged N‐heterotriangulene, a triphenylamine with sulfur atoms bridging the ortho‐positions, was synthesized by a sequence of regioselective sulfenylation with phthalimidesulfenyl chloride followed by Lewis acid‐catalyzed electrophilic cyclization. X‐ray crystallography revealed a saddle‐shaped geometry of the polycyclic scaffold. UV/Vis absorption spectroscopy and cyclic voltammetry were used to characterize the optoelectronic properties. The title compound is a particularly strong electron donor forming a perfectly stable radical cation, which was analyzed by electron paramagnetic resonance spectroscopy and X‐ray crystallography. The electron donor properties were further highlighted by the formation of crystalline donor‐acceptor complexes with strong cyano‐based acceptors.

A Trithia-Bridged N-Heterotriangulene: The Hitherto Missing Electron Donor.

The very first representative of trithia-bridged N-heterotriangulene, a triphenylamine with sulfur atoms bridging the ortho-positions, was synthesized by a sequence of regioselective sulfenylation with phthalimidesulfenyl chloride followed by Lewis acid-catalyzed electrophilic cyclization. X-ray crystallography revealed a saddle-shaped geometry of the polycyclic scaffold. UV/Vis absorption spectroscopy and cyclic voltammetry were used to characterize the optoelectronic properties. The title compound is a particularly strong electron donor forming a perfectly stable radical cation, which was analyzed by electron paramagnetic resonance spectroscopy and X-ray crystallography. The electron donor properties were further highlighted by the formation of crystalline donor-acceptor complexes with strong cyano-based acceptors.

Influence of scaffold geometry on the degradation rate of 3D printed polylactic acid bone scaffold.

Fabricating scaffolds using three-dimensional (3D) printing is an emerging approach in tissue engineering (TE), where filaments with a controlled arrangement are printed. Using fused deposition modeling in bone replacement enables the simulation of bone structure. However, the microenvironment created by the scaffold must meet specific requirements. These requirements aim to create an environment that promotes adhesion, proliferation, differentiation, and cell migration. One of the challenges in creating polylactic acid scaffolds is controlling the degradation rate to match the target tissue. This study investigates the degradation of scaffolds with different geometries and the relationship between scaffolds' geometry and degradation rate. These scaffolds are made of polylactic acid and prepared using 3D printing. The lattice geometry was exposed to acidic media with varying pH levels for 1month, and pH2 was selected for all geometries for further investigation. The five selected geometries were then immersed in the desired acid for 2months, and measurements were taken for wet weight, dry weight, morphology, molecular weight, and crystallinity during degradation. The results showed that the hexagonal sample had a 1.5% increase in wet weight, and the gyroid sample had a 1.2% increase, indicating that the wavy shapes had a higher fluid-holding capacity. The degradation analysis indicated that the hexagonal geometry had accelerated degradation compared to the other geometries. Based on these findings, it can be concluded that filament separation not only results in rapid cooling and prevents the recovery of the crystalline arrangement but also increases the surface area to volume ratio, allowing for more acid penetration and faster degradation. Finally, mechanical properties and in vitro evaluation were assessed for three selected geometries. On the 60th day, the hexagonal scaffold had the highest elastic modulus value of 105 ± 0.45MPa, while the gyroid scaffold had the lowest value of 58.8 ± 0.40MPa. The lattice scaffold had the highest amount of cell attachment, with 210.88 ± 0.35 cells surviving after 24 hours and 94.01 ± 0.18 cells surviving after 72 hours. These high viability rates indicate that the three scaffolds with the selected geometries are suitable for promoting cell growth.

On-chip fabrication of tailored 3D hydrogel scaffolds to model cancer cell invasion and interaction with endothelial cells.

The high mortality associated with certain cancers can be attributed to the invasive nature of the tumor cells. Yet, the complexity of studying invasion hinders our understanding of how the tumor spreads. This work presents a microengineered three-dimensional (3D) in vitro model for studying cancer cell invasion and interaction with endothelial cells. The model was generated by printing a biomimetic hydrogel scaffold directly on a chip using 2-photon polymerization that simulates the brain's extracellular matrix. The scaffold's geometry was specifically designed to facilitate the growth of a continuous layer of endothelial cells on one side, while also allowing for the introduction of tumor cells on the other side. This arrangement confines the cells spatially and enables in situ microscopy of the cancer cells as they invade the hydrogel scaffold and interact with the endothelial layer. We examined the impact of 3D printing parameters on the hydrogel's physical properties and used patient derived glioblastoma cells to study their effect on cell invasion. Notably, the tumor cells tended to infiltrate faster when an endothelial cell barrier was present. The potential for adjusting the hydrogel scaffold's properties, coupled with the capability for real-time observation of tumor-endothelial cell interactions, offers a platform for studying tumor invasion and tumor-endothelial cell interactions.

A Simplified Mathematical Model for Cell Proliferation in a Tissue-Engineering Scaffold.

While the effects of external factors like fluid mechanical forces and scaffold geometry on tissue growth have been extensively studied, the influence of cell behavior-particularly nutrient consumption and depletion within the scaffold-has received less attention. Incorporating such factors into mathematical models allows for a more comprehensive understanding of tissue-engineering processes. This work presents a comprehensive continuum model for cell proliferation within two-dimensional tissue-engineering scaffolds. Through mathematical modeling and asymptotic analysis based on the small aspect ratio of the scaffolds, the study aims to reduce computational burdens and solve mathematical models for tissue growth within porous scaffolds. The model incorporates fluid dynamics of nutrient feed flow, nutrient transport, cell concentration, and tissue growth, considering the evolving scaffold porosity due to cell proliferation, with the crux of the work establishing the ideal pore shape for channels within the tissue-engineering scaffold to obtain the maximum tissue growth. We investigate scaffolds with specific two-dimensional initial porosity profiles, and our results show that scaffolds which are uniformly graded in porosity throughout their depth promote more tissue growth.

Quasi-static and fatigue performance of Ti-6Al-4V triply periodic minimal surface scaffolds manufactured via laser powder bed fusion for hard-tissue engineering

Additive manufacturing of Triply Periodic Minimum Surface scaffolds offers a promising solution for bone defect restoration and an alternative to traditional implants in hard tissue engineering. This study investigates the quasi-static and tension-compression cyclic properties of laser powder bed fusion-produced Ti6Al4V lattice-based architectures, specifically gyroid and diamond scaffold geometries. The scaffolds were designed to emulate the mechanical properties of bones, with pore sizes (650–1000 μm) suitable for tissue engineering, and porosities ranging from to 30–60 %. The quasi-static tensile properties of the stiffness and yield strength were characterized experimentally and validated numerically using the finite-element method. The results showed that the stiffness and tensile strength of the scaffolds were comparable to those of cortical bone, reducing the risk of scaffold failure. The influence of post-processing by heat treatment and hot isostatic pressing resulted in an increased fatigue limit and ductility compared with non-treated materials reported in the literature. Fractography analysis revealed that fatigue fracture initiation occurred at the outer surface of the skeleton structures, where the maximum tensile stress was concentrated. These findings support the assumption that the designed scaffolds accurately replicate the mechanical and fatigue characteristics of cortical bone, offering suitable alternatives for bone replacement and orthopedic implants.

Stereolithography-assisted sodium alginate-collagen hydrogel scaffold with molded internal channels

Fabricating internal vascular networks within a hydrogel scaffold is essential for facilitating the supply of nutrients, oxygen, and metabolism exchange required by the encapsulated cells. The challenges in current hydrogel scaffold fabrication involve the difficulty of building adequate internal channels, poor scaffold geometry precision, and low cell viability caused by the fabrication process and polymer material properties. Stereolithography (SLA) stands out as a 3D printing technique distinguished by its superior production efficiency, advanced precision, and remarkable resolution in crafting intricate custom geometries. These attributes establish it as an innovative approach for templates in scaffold fabrication, potentially surpassing the fused deposition modeling (FDM)-based template strategy. Meanwhile, it exerts less shear stress on the cells compared to the direct bioprinting process. This novel strategy enables the fabrication of hydrogel vascular structure within the precision of 500 µm in both channel diameter and wall thickness. In this paper, various sodium alginate and collagen (SA-Col) composite hydrogels with varying collagen concentrations have been investigated to identify the optimal ratio for fabricating hydrogel scaffolds with channels.

An Integrated Dual-Layer Heterogeneous Polycaprolactone Scaffold Promotes Oral Mucosal Wound Healing through Inhibiting Bacterial Adhesion and Mediating HGF-1 Behavior.

Recently, the high incidence of oral mucosal defects and the subsequent functional impairments have attracted widespread attention. Controlling scaffold geometry pattern has been proposed as a strategy to promote cell behavior and facilitate soft tissue repair. In this study, we innovatively construct an integrated dual-layer heterogeneous polycaprolactone (PCL) scaffold using melt electrowriting (MEW) technology. The outer layer was disordered, while the inner layer featured oriented fiber patterns: parallel (P-par), rhombic (P-rhomb), and square (P-sq). Our findings revealed that the P-rhomb and P-sq scaffolds exhibited superior surface wettability, roughness, and tensile strength compared to the pure disordered PCL scaffolds (P) and P-par. Compared to the commercial collagen membranes, the outer layer of PCL can effectively inhibit bacterial adhesion and biofilm formation. Furthermore, the P-rhomb and P-sq groups demonstrated higher gene and protein expression levels related to cell adhesion and cell migration rates than did the P and P-par groups. Among them, P-sq plays an important role in inducing the differentiation of gingival fibroblasts into myofibroblasts rich in α-smooth muscle actin (α-SMA). Additionally, P-sq could reduce inflammation, promote epithelial regeneration, and accelerate wound healing when used in full-thickness oral mucosal defects in rabbits. Overall, the integrated dual-layer heterogeneous PCL scaffold fabricated by MEW technology effectively inhibited bacterial adhesion and guided tissue regeneration, offering advantages for clinical translation and large-scale production. This promising material holds important potential for treating full-thickness mucosal defects in a bacteria-rich oral environments.

A versatile 5-axis melt electrowriting platform for unprecedented design freedom of 3D fibrous scaffolds

Melt electrowriting (MEW) is an electrohydrodynamic additive manufacturing technology for the fabrication of precise microfiber scaffold architectures but is so far restricted to printing onto simple collector geometries and, therefore, constrained in design freedom. This is due to current limitations in print head designs, kinematic systems, and software to generate motion commands. We address these aspects by upgrading a low-cost 5-axis kinematic system and complete it with a custom-designed print head and a collector fabrication workflow to enable collision-free MEW onto arbitrarily shaped multicurvature 3D geometries. A universal graphical approach enables generation of Gcodes customized to the 5-axis MEW requirements. The print head is validated by producing polycaprolactone fibers with diameters ranging from 5.6 ± 1.7 µm to 50.7 ± 5.5 µm and by writing both linear and non-linear fiber patterns onto collectors featuring concave and convex surfaces. Stable printing conditions are achieved by continuously reorienting the print head and collector surface perpendicular to each other at a constant working distance. Ultimately, we demonstrate the system’s potential in shaping anatomically relevant scaffold geometries by stacking microfibers onto collectors resembling a trileaflet valve and a bifurcating vessel. This multi-axis MEW platform unlocks exciting avenues towards unprecedented design freedom for MEW scaffolds.

Accessible melt electrowriting three-dimensional printer for fabricating high-precision scaffolds

Melt electrowriting (MEW) is an established 3D printing technology for producing high-resolution scaffolds for tissue engineering. Commercial MEW devices are expensive while having limited processing capabilities, which limits their accessibility and widespread use. Herein, we report an easy-to-assemble and inexpensive ($2000) MEW printer based on an off-the-shelf automated dispensing machine with user-friendly controls and a custom-designed MEW printhead capable of printing polymers with a melting point of up to 260 °C. Using the gold standard printable material, poly(ε-caprolactone) (PCL), this printer can deposit 3 μm diameter fibers at 50 μm spacing, demonstrating that the highest quality of scaffolds can be produced on the low-cost MEW system. Stability in printing is improved by maintaining a relative humidity of 30–35 %, ensuring nozzle conditions are optimal, and minimizing bubbles in the polymer melt. Key printing parameters such as a 3 kV applied voltage, 75 °C heating temperature, 100 kPa air pressure, and 30 G nozzle size are critical for high accuracy, achieving 100 μm fiber spacing and layer stacking up to 30 layers without fiber breakage. The printer effectively creates complex scaffold geometries, showcasing its precision and suitability for advanced tissue engineering applications. Overall, this study promotes low-cost MEW technology by establishing a guideline on building an accessible but capable MEW printer and providing a crucial experimental foundation toward achieving high-accuracy scaffolds.

3D-printed gradient scaffolds for osteochondral defects: Current status and perspectives.

Articular osteochondral defects are quite common in clinical practice, and tissue engineering techniques can offer a promising therapeutic option to address this issue.The articular osteochondral unit comprises hyaline cartilage, calcified cartilage zone (CCZ), and subchondral bone.As the interface layer of articular cartilage and bone, the CCZ plays an essentialpart in stress transmission and microenvironmental regulation.Osteochondral scaffolds with the interface structure for defect repair are the future direction of tissue engineering. Three-dimensional (3D) printing has the advantages of speed, precision, and personalized customization, which can satisfy the requirements of irregular geometry, differentiated composition, and multilayered structure of articular osteochondral scaffolds with boundary layer structure. This paper summarizes the anatomy, physiology, pathology, and restoration mechanisms of the articular osteochondral unit, and reviews the necessity for a boundary layer structure in osteochondral tissue engineering scaffolds and the strategy for constructing the scaffolds using 3D printing. In the future, we should not only strengthen the basic research on osteochondral structural units, but also actively explore the application of 3D printing technology in osteochondral tissue engineering. This will enable better functional and structural bionics of the scaffold, which ultimately improve the repair of osteochondral defects caused by various diseases.

Bicyclic Pyrrolidine Inhibitors of Toxoplasma gondii Phenylalanine t-RNA Synthetase with Antiparasitic Potency In Vitro and Brain Exposure.

Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular, to achieve optimal exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency and ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analogue series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.

Porosity and surface curvature effects on the permeability and wall shear stress of trabecular bone: Guidelines for biomimetic scaffolds for bone repair

Scaffolds are an essential component of bone tissue engineering to provide support and create a physiological environment for cells. Biomimetic scaffolds are a promising approach to fulfill the requirements. Bone allografts are widely used scaffolds due to their mechanical and structural characteristics. The scaffold geometry is well known to be an important determinant of induced mechanical stimulation felt by the cells. However, the impact of allograft geometry on permeability and wall shear stress distribution is not well understood. This information is essential for designing biomimetic scaffolds that provide a suitable environment for cells to proliferate and differentiate. The present study investigates the effect of geometry on the permeability and wall shear stress of bone allografts at both macroscopic and microscopic scales. Our results concluded that the wall shear stress was strongly correlated with the porosity of the allograft. The level of wall shear stress at a local scale was also determined by the surface curvature characteristics. The results of this study can serve as a guideline for future biomimetic scaffold designs that provide a mechanical environment favorable for osteogenesis and bone repair.

Design, In Vitro Evaluation and In Vivo Biocompatibility of Additive Manufacturing Three-Dimensional Printing of β beta-Tricalcium Phosphate Scaffolds for Bone Regeneration

The reconstruction of bone deficiencies remains a challenge due to the limitations of autologous bone grafting. The objective of this study is to evaluate the bone regeneration efficacy of additive manufacturing of tricalcium phosphate (TCP) implants using lithography-based ceramic manufacturing (LCM). LCM uses LithaBone TCP 300 slurry for 3D printing, producing cylindrical scaffolds. Four models of internal scaffold geometry were developed and compared. The in vitro studies included cell culture, differentiation, seeding, morphological studies and detection of early osteogenesis. The in vivo studies involved 42 Wistar rats divided into four groups (control, membrane, scaffold (TCP) and membrane with TCP). In each animal, unilateral right mandibular defects with a total thickness of 5 mm were surgically performed. The animals were sacrificed 3 and 6 months after surgery. Bone neoformation was evaluated by conventional histology, radiology, and micro-CT. Model A (spheres with intersecting and aligned arrays) showed higher penetration and interconnection. Histological and radiological analysis by micro-CT revealed increased bone formation in the grafted groups, especially when combined with a membrane. Our innovative 3D printing technology, combined with precise scaffold design and efficient cleaning, shows potential for bone regeneration. However, further refinement of the technique and long-term clinical studies are crucial to establish the safety and efficacy of these advanced 3D printed scaffolds in human patients.

Indenoannulated Tridecacyclene: An All-Carbon Seven-Stage Redox-Amphoter.

We disclose an indenoannulated tridecacyclene comprising a central cyclooctatetraene moiety with multiple adjacent pentagonal rings which is accessible in a concise synthetic sequence. The saddle-shaped geometry of the non-benzenoid polycyclic scaffold and its unique packing behavior in the solid state were characterized by X-ray crystallography. In electrochemical studies, the compound undergoes seven reversible redox events comprising five reductions and two oxidations. The dicationic and dianionic species obtained by chemical oxidation and reduction, respectively, were characterized spectroscopically in solution. Density functional theory calculations were applied to provide insights into aromaticity evolution in the respective charged species, highlighting the beneficial effect of the non-benzenoid moieties on charge stabilization.

Rationally Designing the Supramolecular Interfaces of Nanoparticle Superlattices with Multivalent Polymers.

In supramolecular materials, multiple weak binding groups can act as a single collective unit when confined to a localized volume, thereby producing strong but dynamic bonds between material building blocks. This principle of multivalency provides a versatile means of controlling material assembly, as both the number and the type of supramolecular moieties become design handles to modulate the strength of intermolecular interactions. However, in materials with building blocks significantly larger than individual supramolecular moieties (e.g., polymer or nanoparticle scaffolds), the degree of multivalency is difficult to predict or control, as sufficiently large scaffolds inherently preclude separated supramolecular moieties from interacting. Because molecular models commonly used to examine supramolecular interactions are intrinsically unable to examine any trends or emergent behaviors that arise due to nanoscale scaffold geometry, our understanding of the thermodynamics of these massively multivalent systems remains limited. Here we address this challenge via the coassembly of polymer-grafted nanoparticles and multivalent polymers, systematically examining how multivalent scaffold size, shape, and spacing affect their collective thermodynamics. Investigating the interplay of polymer structure and supramolecular group stoichiometry reveals complicated but rationally describable trends that demonstrate how the supramolecular scaffold design can modulate the strength of multivalent interactions. This approach to self-assembled supramolecular materials thus allows for the manipulation of polymer-nanoparticle composites with controlled thermal stability, nanoparticle organization, and tailored meso- to microscopic structures. The sophisticated control of multivalent thermodynamics through precise modulation of the nanoscale scaffold geometry represents a significant advance in the ability to rationally design complex hierarchically structured materials via self-assembly.

SpyMask enables combinatorial assembly of bispecific binders

Bispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry.