Supramolecular Hydrolase Mimics in Equilibrium and Kinetically Trapped States.

The folding of proteins into intricate three-dimensional structures to achieve biological functions, such as catalysis, is governed by both kinetic and thermodynamic controls. The quest to design artificial enzymes using minimalist peptides seeks to emulate supramolecular structures existing in a catalytically active state. Drawing inspiration from the nuanced process of protein folding, our study explores the enzyme-like activity of amphiphilic peptide nanosystems in both equilibrium and non-equilibrium states, featuring the formation of supramolecular nanofibrils and nanosheets. In contrast to thermodynamically stable nanosheets, the kinetically trapped nanofibrils exhibit dynamic characteristics (e.g., rapid molecular exchange and relatively weak intermolecular packing), resulting in a higher hydrolase-mimicking activity. We emphasize that a supramolecular microenvironment characterized by an optimal local polarity, microviscosity, and ß-sheet hydrogen bonding is conducive to both substrate binding and ester bond hydrolysis. Our work underscores the pivotal role of both thermodynamic and kinetic control in impacting biomimetic catalysis and sheds a light on the development of artificial enzymes.

Strategies to Control or Mimic Growth Factor Activity for Bone, Cartilage, and Osteochondral Tissue Engineering.

Growth factors play a critical role in tissue repair and regeneration. However, their clinical success is limited by their low stability, short half-life, and rapid diffusion from the delivery site. Supraphysiological growth factor concentrations are often required to demonstrate efficacy but can lead to adverse reactions, such as inflammatory complications and increased cancer risk. These issues have motivated the development of delivery systems that enable sustained release and controlled presentation of growth factors. This review specifically focuses on bioconjugation strategies to enhance growth factor activity for bone, cartilage, and osteochondral applications. We describe approaches to localize growth factors using noncovalent and covalent methods, bind growth factors via peptides, and mimic growth factor function with mimetic peptide sequences. We also discuss emerging and future directions to control spatiotemporal growth factor delivery to improve functional tissue repair and regeneration.

Interlayer bonding strength of 3D printed PEEK specimens.

Recent advances in extrusion-based filament 3D printing technology enable the processability of high-performance polymers. Poly(ether ether ketone) (PEEK) is an important group of high-performance polymer that has been widely used in aerospace, automotive, and biomedical applications. The interlayer bonding strength of 3D printed PEEK is crucial for load-bearing applications, yet studies on 3D printed PEEK are sparse due to processing challenges. In this study, the three-point flexural test is used to study the interlayer bonding strength of 3D-printed PEEK specimens with respect to the printing process parameters, including nozzle temperature, print speed, layer height, and wait-time. A design of experiment (DOE) approach is developed to study correlations between printing parameters and the end-use properties, including flexural stress (σf) and strain at break (εf), flexural modulus (Ef), and crystallinity (χ). Our results show that the nozzle temperature, layer height, and wait-time significantly affect the interlayer bonding strength, with nozzle temperature being the most influential parameter to enhance interlayer bonding strength indicated by a significant increase in σf, εf, and χ. Thermal annealing post-printing is shown to increase the degree of χ and Ef, yet its effect on interlayer bonding strength is minimal, indicating that the interlayer bonding strength is primarily determined during the printing process. This study demonstrates the use of a three-point flexural test integrated with a versatile and robust DOE approach to study the interlayer bonding strength of PEEK to reduce product development time while improving mechanical properties.

Plasmonic Chirality Imprinting on Nucleobase-Displaying Supramolecular Nanohelices by Metal-Nucleobase Recognition.

Supramolecular self-assembly is an important process that enables the conception of complex structures mimicking biological motifs. Herein, we constructed helical fibrils through chiral self-assembly of nucleobase-peptide conjugates (NPCs), where achiral nucleobases are helically displayed on the surface of fibrils, comparable to polymerized nucleic acids. Selective binding between DNA and the NPC fibrils was observed with fluorescence polarization. Taking advantage of metal-nucleobase recognition, we highlight the possibility of deposition/assembly of plasmonic nanoparticles onto the fibrillar constructs. In this approach, the supramolecular chirality of NPCs can be adaptively imparted to metallic nanoparticles, covering them to generate structures with plasmonic chirality that exhibit significantly improved colloidal stability. The self-assembly of rationally designed NPCs into nanohelices is a promising way to engineer complex, optically diverse nucleobase-derived nanomaterials.

Modular and Versatile Spatial Functionalization of Tissue Engineering Scaffolds through Fiber-Initiated Controlled Radical Polymerization.

Native tissues are typically heterogeneous and hierarchically organized, and generating scaffolds that can mimic these properties is critical for tissue engineering applications. By uniquely combining controlled radical polymerization (CRP), end-functionalization of polymers, and advanced electrospinning techniques, a modular and versatile approach is introduced to generate scaffolds with spatially organized functionality. Poly-ε-caprolactone is end functionalized with either a polymerization-initiating group or a cell-binding peptide motif cyclic Arg-Gly-Asp-Ser (cRGDS), and are each sequentially electrospun to produce zonally discrete bilayers within a continuous fiber scaffold. The polymerization-initiating group is then used to graft an antifouling polymer bottlebrush based on poly(ethylene glycol) from the fiber surface using CRP exclusively within one bilayer of the scaffold. The ability to include additional multifunctionality during CRP is showcased by integrating a biotinylated monomer unit into the polymerization step allowing postmodification of the scaffold with streptavidin-coupled moieties. These combined processing techniques result in an effective bilayered and dual-functionality scaffold with a cell-adhesive surface and an opposing antifouling non-cell-adhesive surface in zonally specific regions across the thickness of the scaffold, demonstrated through fluorescent labelling and cell adhesion studies. This modular and versatile approach combines strategies to produce scaffolds with tailorable properties for many applications in tissue engineering and regenerative medicine.

Electrostatic control of structure in self-assembled membranes.

Self-assembling peptide amphiphiles (PAs) can form hierarchically ordered membranes when brought in contact with aqueous polyelectrolytes of the opposite charge by rapidly creating a diffusion barrier composed of filamentous nanostructures parallel to the plane of the incipient membrane. Following this event, osmotic forces and charge complexation template nanofiber growth perpendicular to the plane of the membrane in a dynamic self-assembly process. In this work, we show that this hierarchical structure requires massive interfacial aggregation of PA molecules, suggesting the importance of rapid diffusion barrier formation. Strong PA aggregation is induced here through the use of heparin-binding PAs with heparin and also with polyelectrolytes of varying charge density. Small angle X-ray scattering shows that in the case of weak PA-polyelectrolyte interaction, membranes formed display a cubic phase ordering on the nanoscale that likely results from clusters of PA nanostructures surrounded by polyelectrolyte chains.

Self-assembly of collagen building blocks guided by electric fields.

Show me the way: protein building blocks are programmed to assemble hierarchically and yield a defined fiber morphology of micrometric length and precise nanometric diameter. The key step of this method is to align the building blocks with an AC field prior to assembly. The resulting protein nanofibers are straightforwardly integrated with the circuitry for potential applications in bionanotechnology.

Immunomodulatory Strategies for Cartilage Regeneration in Osteoarthritis.

Osteoarthritis (OA) is the most prevalent musculoskeletal disorder and a leading cause of disability globally. Although many efforts have been made to treat this condition, current tissue engineering (TE) and regenerative medicine strategies fail to address the inflammatory tissue environment that leads to the rapid progression of the disease and prevents cartilage tissue formation. First, this review addresses in detail the current anti-inflammatory therapies for OA with a special emphasis on pharmacological approaches, gene therapy, and mesenchymal stromal cell (MSC) intra-articular administration, and discusses the reasons behind the limited clinical success of these approaches at enabling cartilage regeneration. Then, we analyze the state-of-the-art TE strategies and how they can be improved by incorporating immunomodulatory capabilities such as the optimization of biomaterial composition, porosity and geometry, and the loading of anti-inflammatory molecules within an engineered structure. Finally, the review discusses the future directions for the new generation of TE strategies for OA treatment, specifically focusing on the spatiotemporal modulation of anti-inflammatory agent presentation to allow for tailored patient-specific therapies. Impact statement Osteoarthritis (OA) is a prevalent and debilitating musculoskeletal disorder affecting millions worldwide. Despite significant advancements in regenerative medicine and tissue engineering (TE), mitigating inflammation while simultaneously promoting cartilage tissue regeneration in OA remains elusive. In this review article, we discuss current anti-inflammatory therapies and explore their potential synergy with cutting-edge cartilage TE strategies, with a special focus on novel spatiotemporal and patient-specific anti-inflammatory strategies.

Solvent-cast 3D printing with molecular weight polymer blends to decouple effects of scaffold architecture and mechanical properties on mesenchymal stromal cell fate.

The biochemical and physical properties of a scaffold can be tailored to elicit specific cellular responses. However, it is challenging to decouple their individual effects on cell-material interactions. Here, we solvent-cast 3D printed different ratios of high and low molecular weight (MW) poly(caprolactone) (PCL) to fabricate scaffolds with significantly different stiffnesses without affecting other properties. Ink viscosity was used to match processing conditions between inks and generate scaffolds with the same surface chemistry, crystallinity, filament diameter, and architecture. Increasing the ratio of low MW PCL resulted in a significant decrease in modulus. Scaffold modulus did not affect human mesenchymal stromal cell (hMSC) differentiation under osteogenic conditions. However, hMSC response was significantly affected by scaffold stiffness in chondrogenic media. Low stiffness promoted more stable chondrogenesis whereas high stiffness drove hMSC progression toward hypertrophy. These data illustrate how this versatile platform can be used to independently modify biochemical and physical cues in a single scaffold to synergistically enhance desired cellular response.

Characterizing properties of scaffolds 3D printed with peptide-polymer conjugates.

Three-dimensional (3D) printing is a popular biomaterials fabrication technique because it enables scaffold composition and architecture to be tuned for different applications. Modifying these properties can also alter mechanical properties, making it challenging to decouple biochemical and physical properties. In this study, inks containing peptide-poly(caprolactone) (PCL) conjugates were solvent-cast 3D printed to create peptide-functionalized scaffolds. We characterized how different concentrations of hyaluronic acid-binding (HAbind-PCL) or mineralizing (E3-PCL) conjugates influenced properties of the resulting 3D-printed constructs. The peptide sequences CGGGRYPISRPRKR (HAbind-PCL; positively charged) and CGGGAAAEEE (E3-PCL; negatively charged) enabled us to evaluate how conjugate chemistry, charge, and concentration affected 3D-printed architecture, conjugate location, and mechanical properties. For both HAbind-PCL and E3-PCL, conjugate addition did not affect ink viscosity, filament diameter, scaffold architecture, or scaffold compressive modulus. Increasing conjugate concentration in the ink prior to printing correlated with an increase in peptide concentration on the scaffold surface. Interestingly, conjugate type affected final conjugate location within the 3D-printed filament cross-section. HAbind-PCL conjugates remained within the filament bulk while E3-PCL conjugates were located closer to the filament surface. E3-PCL at all concentrations did not affect mechanical properties, but an intermediate HAbind-PCL concentration resulted in a moderate decrease in filament tensile modulus. These data suggest final conjugate location within the filament bulk may influence mechanical properties. However, no significant differences were observed between PCL filaments printed without conjugates and filaments printed with higher HAbind-PCL concentrations. These results demonstrate that this 3D printing platform can be used to functionalize the surface without significant changes to the physical properties of the scaffold. The downstream potential of this strategy will enable decoupling of biochemical and physical properties to fine-tune cellular responses and support functional tissue regeneration.

Spatial organization of biochemical cues in 3D-printed scaffolds to guide osteochondral tissue engineering.

Functional repair of osteochondral (OC) tissue remains challenging because the transition from bone to cartilage presents gradients in biochemical and physical properties necessary for joint function. Osteochondral regeneration requires strategies that restore the spatial composition and organization found in the native tissue. Several biomaterial approaches have been developed to guide chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). These strategies can be combined with 3D printing, which has emerged as a useful tool to produce tunable, continuous scaffolds functionalized with bioactive cues. However, functionalization often includes one or more post-fabrication processing steps, which can lead to unwanted side effects and often produce biomaterials with homogeneously distributed chemistries. To address these challenges, surface functionalization can be achieved in a single step by solvent-cast 3D printing peptide-functionalized polymers. Peptide-poly(caprolactone) (PCL) conjugates were synthesized bearing hyaluronic acid (HA)-binding (HAbind-PCL) or mineralizing (E3-PCL) peptides, which have been shown to promote hMSC chondrogenesis or osteogenesis, respectively. This 3D printing strategy enables unprecedented control of surface peptide presentation and spatial organization within a continuous construct. Scaffolds presenting both cartilage-promoting and bone-promoting peptides had a synergistic effect that enhanced hMSC chondrogenic and osteogenic differentiation in the absence of differentiation factors compared to scaffolds without peptides or only one peptide. Furthermore, multi-peptide organization significantly influenced hMSC response. Scaffolds presenting HAbind and E3 peptides in discrete opposing zones promoted hMSC osteogenic behavior. In contrast, presenting both peptides homogeneously throughout the scaffolds drove hMSC differentiation towards a mixed population of articular and hypertrophic chondrocytes. These significant results indicated that hMSC behavior was driven by dual-peptide presentation and organization. The downstream potential of this platform is the ability to fabricate biomaterials with spatially controlled biochemical cues to guide functional tissue regeneration without the need for differentiation factors.

Fabricating spatially functionalized 3D-printed scaffolds for osteochondral tissue engineering.

Three-dimensional (3D) printing of biodegradable polymers has rapidly become a popular approach to create scaffolds for tissue engineering. This technique enables fabrication of complex architectures and layer-by-layer spatial control of multiple components with high resolution. The resulting scaffolds can also present distinct chemical groups or bioactive cues on the surface to guide cell behavior. However, surface functionalization often includes one or more post-fabrication processing steps, which typically produce biomaterials with homogeneously distributed chemistries that fail to mimic the biochemical organization found in native tissues. As an alternative, our laboratory developed a novel method that combines solvent-cast 3D printing with peptide-polymer conjugates to spatially present multiple biochemical cues in a single scaffold without requiring post-fabrication modification. Here, we describe a detailed, stepwise protocol to fabricate peptide-functionalized scaffolds and characterize their physical architecture and biochemical spatial organization. We used these 3D-printed scaffolds to direct human mesenchymal stem cell differentiation and osteochondral tissue formation by controlling the spatial presentation of cartilage-promoting and bone-promoting peptides. This protocol also describes how to seed scaffolds and evaluate matrix deposition driven by peptide organization.

3D printing with peptide-polymer conjugates for single-step fabrication of spatially functionalized scaffolds.

Biodegradable polymer-based scaffolds are widely used to provide support during early stages of regeneration and can be functionalized with various chemical groups or bioactive cues to promote desired cellular behavior. However, these scaffolds are often modified post-fabrication, which can lead to undesired changes and homogeneously distributed chemistries that fail to mimic the spatial biochemical organization found in native tissues. To address these challenges, surface functionalization can be achieved by 3D printing with pre-functionalized biodegradable polymers, such as peptide-modified polymer conjugates, to control the deposition of preferred chemistries. Peptide-PCL conjugates were synthesized with the canonical cell adhesion peptide motif RGDS or its negative control RGES and 3D printed into scaffolds displaying one or both peptides. The peptides were also modified with bioorthogonal groups, biotin and azide, to visualize peptide concentration and location by labeling with complementary fluorophores. Peptide concentration on the scaffold surface increased with increasing peptide-PCL conjugate concentration added to the ink prior to 3D printing, and scaffolds printed with the highest RGDS(biotin)-PCL concentrations showed a significant increase in NIH3T3 fibroblast adhesion. To demonstrate spatial control of peptide functionalization, multiple printer heads were used to print both peptide-PCL conjugates into the same construct in alternating patterns. Cells preferentially attached and spread on RGDS(biotin)-PCL fibers compared to RGES(azide)-PCL fibers, illustrating how spatial functionalization can be used to influence local cell behavior within a single biomaterial. This presents a versatile platform to generate multifunctional biomaterials that can mimic the biochemical organization found in native tissues to support functional regeneration.

Growth factor delivery from self-assembling nanofibers to facilitate islet transplantation.

Recent advances in nanotechnology and molecular self-assembly may provide novel solutions to current cell transplantation deficiencies. Heparin-binding peptide amphiphiles (HBPAs) self-assemble from aqueous media into nanofibers that bind growth factors through interactions with the bioactive polymer heparin. In this report, we demonstrate that delivery of vascular endothelial growth factor and fibroblast growth factor-2 from HBPA scaffolds significantly increases blood vessel density in the mouse omentum over control scaffolds without growth factors (P<0.0005) and significantly enhances islet engraftment. Diabetic recipients transplanted with 250 isologous islets and HBPA scaffolds containing vascular endothelial growth factor/fibroblast growth factor-2 achieved normoglycemia at a higher rate (78%) than control animals receiving identical scaffolds without growth factors (30%; P<0.05) or growth factors alone (20%). These data indicate that the enhanced engraftment can be attributed to specific growth factor effects that were made possible by the delivery mechanism of HBPA nanostructures.

Electrospinning Functionalized Polymers for Use as Tissue Engineering Scaffolds.

Electrospinning polymers is a versatile technique to generate fibrous, three-dimensional scaffolds for tissue engineering applications. Modifying polymers with functional groups prior to electrospinning offers the opportunity to control the spatial presentation of functional groups within the scaffold as well as incorporate multiple bioactive cues. This chapter describes methods to modify poly(ε-caprolactone) (PCL) with peptides and electrospin these peptide-PCL conjugates to functionalize a scaffold surface in a single step. Methods to adapt standard electrospinning setups to create single- or dual-peptide gradients within a single construct are also described.

Multimodal Hydrogel-Based Platform To Deliver and Monitor Cardiac Progenitor/Stem Cell Engraftment.

Retention and survival of transplanted cells are major limitations to the efficacy of regenerative medicine, with short-term paracrine signals being the principal mechanism underlying current cell therapies for heart repair. Consequently, even improvements in short-term durability may have a potential impact on cardiac cell grafting. We have developed a multimodal hydrogel-based platform comprised of a poly(ethylene glycol) network cross-linked with bioactive peptides functionalized with Gd(III) in order to monitor the localization and retention of the hydrogel in vivo by magnetic resonance imaging. In this study, we have tailored the material for cardiac applications through the inclusion of a heparin-binding peptide (HBP) sequence in the cross-linker design and formulated the gel to display mechanical properties resembling those of cardiac tissue. Luciferase-expressing cardiac stem cells (CSC-Luc2) encapsulated within these gels maintained their metabolic activity for up to 14 days in vitro. Encapsulation in the HBP hydrogels improved CSC-Luc2 retention in the mouse myocardium and hind limbs at 3 days by 6.5- and 12- fold, respectively. Thus, this novel heparin-binding based, Gd(III)-tagged hydrogel and CSC-Luc2 platform system demonstrates a tailored, in vivo detectable theranostic cell delivery system that can be implemented to monitor and assess the transplanted material and cell retention.

Peptide-Functionalized Fluorescent Particles for In Situ Detection of Nitric Oxide via Peroxynitrite-Mediated Nitration.

Nitric oxide (NO) is a free radical signaling molecule that plays a crucial role in modulating physiological homeostasis across multiple biological systems. NO dysregulation is linked to the pathogenesis of multiple diseases; therefore, its quantification is important for understanding pathophysiological processes. The detection of NO is challenging, typically limited by its reactive nature and short half-life. Additionally, the presence of interfering analytes and accessibility to biological fluids in the native tissues make the measurement technically challenging and often unreliable. Here, a bio-inspired peptide-based NO sensor is developed, which detects NO-derived oxidants, predominately peroxynitrite-mediated nitration of tyrosine residues. It is demonstrated that these peptide-based NO sensors can detect peroxynitrite-mediated nitration in response to physiological shear stress by endothelial cells in vitro. Using the peptide-conjugated fluorescent particle immunoassay, peroxynitrite-mediated nitration activity with a detection limit of ≈100 × 10-9 m is detected. This study envisions that the NO detection platform can be applied to a multitude of applications including monitoring of NO activity in healthy and diseased tissues, localized detection of NO production of specific cells, and cell-based/therapeutic screening of peroxynitrite levels to monitor pronitroxidative stress in biological samples.

Enhanced articular cartilage by human mesenchymal stem cells in enzymatically mediated transiently RGDS-functionalized collagen-mimetic hydrogels.

Recapitulation of the articular cartilage microenvironment for regenerative medicine applications faces significant challenges due to the complex and dynamic biochemical and biomechanical nature of native tissue. Towards the goal of biomaterial designs that enable the temporal presentation of bioactive sequences, recombinant bacterial collagens such as Streptococcal collagen-like 2 (Scl2) proteins can be employed to incorporate multiple specific bioactive and biodegradable peptide motifs into a single construct. Here, we first modified the backbone of Scl2 with glycosaminoglycan-binding peptides and cross-linked the modified Scl2 into hydrogels via matrix metalloproteinase 7 (MMP7)-cleavable or non-cleavable scrambled peptides. The cross-linkers were further functionalized with a tethered RGDS peptide creating a system whereby the release from an MMP7-cleavable hydrogel could be compared to a system where release is not possible. The release of the RGDS peptide from the degradable hydrogels led to significantly enhanced expression of collagen type II (3.9-fold increase), aggrecan (7.6-fold increase), and SOX9 (5.2-fold increase) by human mesenchymal stem cells (hMSCs) undergoing chondrogenesis, as well as greater extracellular matrix accumulation compared to non-degradable hydrogels (collagen type II; 3.2-fold increase, aggrecan; 4-fold increase, SOX9; 2.8-fold increase). Hydrogels containing a low concentration of the RGDS peptide displayed significantly decreased collagen type I and X gene expression profiles, suggesting a major advantage over either hydrogels functionalized with a higher RGDS peptide concentration, or non-degradable hydrogels, in promoting an articular cartilage phenotype. These highly versatile Scl2 hydrogels can be further manipulated to improve specific elements of the chondrogenic response by hMSCs, through the introduction of additional bioactive and/or biodegradable motifs. As such, these hydrogels have the possibility to be used for other applications in tissue engineering. STATEMENT OF SIGNIFICANCE: Recapitulating aspects of the native tissue biochemical microenvironment faces significant challenges in regenerative medicine and tissue engineering due to the complex and dynamic nature of the tissue. The ability to take advantage of, mimic, and modulate cell-mediated processes within novel naturally-derived hydrogels is of great interest in the field of biomaterials to generate constructs that more closely resemble the biochemical microenvironment and functions of native biological tissues such as articular cartilage. Towards this goal, the temporal presentation of bioactive sequences such as RGDS on the chondrogenic differentiation of human mesenchymal stem cells is considered important as it has been shown to influence the chondrogenic phenotype. Here, a novel and versatile platform to recreate a high degree of biological complexity is proposed, which could also be applicable to other tissue engineering and regenerative medicine applications.

Creating biomaterials with spatially organized functionality.

Biomaterials for tissue engineering provide scaffolds to support cells and guide tissue regeneration. Despite significant advances in biomaterials design and fabrication techniques, engineered tissue constructs remain functionally inferior to native tissues. This is largely due to the inability to recreate the complex and dynamic hierarchical organization of the extracellular matrix components, which is intimately linked to a tissue's biological function. This review discusses current state-of-the-art strategies to control the spatial presentation of physical and biochemical cues within a biomaterial to recapitulate native tissue organization and function.

Controlled Sub-Nanometer Epitope Spacing in a Three-Dimensional Self-Assembled Peptide Hydrogel.

Cells in the body use a variety of mechanisms to ensure the specificity and efficacy of signal transduction. One way that this is achieved is through tight spatial control over the position of different proteins, signaling sequences, and biomolecules within and around cells. For instance, the extracellular matrix protein fibronectin presents RGDS and PHSRN sequences that synergistically bind the α5ß1 integrin when separated by 3.2 nm but are unable to bind when this distance is >5.5 nm.1 Building biomaterials to controllably space different epitopes with subnanometer accuracy in a three-dimensional (3D) hydrogel is challenging. Here, we synthesized peptides that self-assemble into nanofiber hydrogels utilizing the ß-sheet motif, which has a known regular spacing along the peptide backbone. By modifying specific locations along the peptide, we are able to controllably space different epitopes with subnanometer accuracy at distances from 0.7 nm to over 6 nm, which is within the size range of many protein clusters. Endothelial cells encapsulated within hydrogels displaying RGDS and PHSRN in the native 3.2 nm spacing showed a significant upregulation in the expression of the alpha 5 integrin subunit compared to those in hydrogels with a 6.2 nm spacing, demonstrating the physiological relevance of the spacing. Furthermore, after 24 h the cells in hydrogels with the 3.2 nm spacing appeared to be more spread with increased staining for the α5ß1 integrin. This self-assembling peptide system can controllably space multiple epitopes with subnanometer accuracy, demonstrating an exciting platform to study the effects of ligand density and location on cells within a synthetic 3D environment.