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## Section 6.2 – Protein Self-Assembly

Olga Matsarskaia<fnref n="1" />, Frank Schreiber<fnref n="2" /> and Felix Roosen-Runge<fnref n="3" />

**Status** Processes of protein self-assembly are highly relevant in a broad range of research fields linked to soft matter, covering aspects from fundamental understanding of life to everyday applications and advanced biotechnology. While the individual protein molecule already presents a complex folded structure, assembly of proteins frequently governs biological function such as during enzymatic reactions, cyteskeleton formation, viral replication, and condensation of membraneless organelles [1-3]. This central role of protein assembly for life is also reflected in biomedical and pharmaceutical challenges. Examples are administrable (i.e., sufficiently low viscosity) antibody formulations, fibril formation in neurodegenerative diseases, and protein condensation linked to age-related eye cataract. In terms of essential research tools, structure determination of proteins is still mostly dependent on the controlled formation of protein crystals. Biomaterials represent another advanced application of protein assembly – often in a biomimetic spirit – [4], where specific material properties enable biomedical applications such as drug delivery and tissue engineering. Recent approaches based on established soft matter accounts in food science [5-7] aim for controlled assembly of plant proteins to produce sustainable alternatives to meat and dairy products. Besides these advanced applications from the last decades, protein assembly has been exploited in traditional food production (e.g., cheese and bread) for millennia.

As evident from these examples, protein self-assembly in a broader sense does not only imply the formation of selective, specific, and rigid protein structures, but also contributes to a dynamic picture of transient and disordered protein assemblies such as dense condensates which represent a growing research interest due to their role in cellular metabolic pathways. Currently, multiple key questions regarding protein self-assembly remain unsolved. For example, a detailed understanding of how molecular dynamics drives assembly processes is missing. Examples include the role of protein docking in metabolic regulation or responses of proteins to external stimuli, such as allosteric effects. On the level of macroscopic emergent phenomena, the link between protein phase behaviour and nucleation pathways of solid structures such as fibrils and crystals, as well as formation pathways of hydrogels, remain poorly understood. The role of thermodynamic driving forces giving rise to self-organisation, has been increasingly appreciated [2, 9-10].

In this context, a central question is whether control parameters, e.g., solvent conditions, salts, and other macromolecules, could be used to control protein assembly and phase behaviour [2,11,12], with the aim, inter alia, to provide cures to diseases [1] and to enable rational design of biomaterials [13,14].

**Current and Future Challenges** A first challenge to be understanding of protein assembly is its complex and hierarchical character on multiple length scales, ranging from the primary structure of the amino acid sequence, over secondary structure elements and the tertiary structure of protein domains on nanometer size, to the quaternary structure of multi-domain or multi-subunit proteins, and eventually supramolecular assemblies of different topology on micrometer length scale (see Figure 1).

A second challenge is linked to the soft matter nature of proteins: due to their inherent physico-chemical complexity, proteins comprise aspects of colloids, polymers and amphiphiles. Potential

<footnote n="1">¹ Institut Laue-Langevin, Grenoble, France</footnote>

<footnote n="2">² Institute of Applied Physics, University of Tübingen, Germany</footnote>

<footnote n="3">³ Department of Biomedical Science and Biofilms-Research Center for Biointerfaces, University of Malmö, Sweden</footnote>
interaction modes include colloid-like molecular docking of anisotropic protein molecules; induced-fit assembly, i.e. a combined process of unfolding and assembling; and polymer/polyelectrolyte-like transient interactions, but all this for inherently anisotropic and inhomogeneous objects. These manifold interaction modes point toward a third challenge: Proteins not only flexibly respond to environmental changes on the molecular level – some proteins even have the potential to act as molecular motors or enzymatic facilitators of controlled assembly. A quantitative understanding of biological function of protein assembly and the functionality of biomaterials thus needs to go beyond a mere picture of multi-scale structure. Dynamical signatures on multiple relevant time scales (posing a formidable challenge to experimental methodology) as well as kinetic, non-equilibrium aspects are needed for a conclusive mechanistic understanding [8,15].

Studying protein assembly requires an interdisciplinary and multi-technique approach (see Fig. 2). Combining the appropriate techniques and reaching a comprehensive picture is of utmost importance and requires not only deep methodological knowledge, but also careful organization and cross-disciplinary communication. Very practical challenges start from the production and storage of a sufficient amount and purity of relevant proteins. To cover the plethora of processes on different time and length scales, prominent experimental methods include optical spectroscopy, scattering techniques, NMR, and fluorescence approaches [8]. In addition, theoretical models rooted in statistical physics and stochastic processes as well as molecular dynamics and coarse-grained simulations are valuable tools for mechanistic insights and hypothesis generation for experiments. Protein research thus strongly relies on interdisciplinary and integrative collaboration to translate soft matter approaches to biological systems with relevance for health and medicine.

**Advances in Science and Technology to Meet Challenges** The theoretical description of protein phase behaviour in terms of colloidal models has improved over the last years [16], e.g., by introducing patchy interactions matching real properties of the anisotropic protein molecules [17-20], promising more predictive schemes for future applications. In response to the huge interest in condensates of intrinsically disordered proteins (IDPs), theoretical concepts of polymer and colloid physics have been revisited and adapted [2]. These theoretical aspects are complemented by efforts in computer simulations to improve the force-fields for atomistic molecular dynamics simulations of IDPs and develop coarse-grained realistic models which capture the propensities of local secondary structure even for IDPs [21].

On the experimental side, advances in a range of research fields open new opportunities for complementary and comprehensive studies of protein assemblies. FRET provides specific local information on docking processes in dynamic structural ensembles [22]. New trends of NMR methodology enable the structural characterization of large specific protein assemblies [23]. Super-resolution microscopy emerges as a tool to study growth processes from surfaces using time-resolved real-space imaging [24].

In recent years, time-resolved small-angle scattering in combination with stochastic modeling has been used to unravel assembly pathways of proteins, e.g. by comparing the observed time trace of structural signatures during protein crystallization with rate-equation models [25], by using data-driven analysis methods to extract kinetic and structural information on intermediates in viral assembly [26], or exploiting contrast variation to study the dynamic multi-subunit protein structure [27].

Furthermore, even time-resolved measurements using dynamical scattering techniques are becoming applicable to protein solutions due to the recent instrumentation improvements for X-ray Photon Correlation Spectroscopy (XPCS) [28,29] and Quasi-Elastic Neutron Scattering (QENS) [8].
**Concluding Remarks** Overall, it is becoming increasingly obvious that there is a strong trend towards interdisciplinary work and a pronounced "softening" of the boundaries between traditional research disciplines. Indeed, multi-technique and time-resolved characterization of biomaterials and a consistent combination of experiment and theoretical modeling are becoming more and more important. It is safe to assume that these trends will continue in the future.

**Acknowledgements**

The authors gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG), the German Ministry for Education and Research (BMBF), the Crafoord Foundation as well as various large-scale research facilities (in particular Diamond Light Source, the ISIS Neutron and Muon Source, the European Synchrotron Radiation Facility, the Forschungsreaktor München II, the Julich Center for Neutron Science, the Institut Laue-Langevin and Oak Ridge Neutron Laboratory) for beamtime allocation and excellent on-site support.

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<figure><img src="image_50.png" /><figcaption>**Figure 1** Protein assembly starts from multi-level macromolecular folding and extends to multi-scale and hierarchical structures.</figcaption></figure>

Protein assembly -
A multi-modal integrative research field

<figure><img src="image_51.png" /><figcaption>**Figure 2** Protein assembly combines research approaches from a broad research field, integrating experimental techniques with theoretical and computational modeling.</figcaption></figure>
# Section 6.3 – Biosourced soft materials

Marie-Eve Aubin-Tam and Gijsje H. Koenderink

Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2629 HZ Delft, The Netherlands

**Status** Biosourced soft materials consist of soft materials derived from or manufactured (at least partly) by living organisms. They fall under the general term ‘biomaterials’, which usually refers to materials in three categories: [1] Biosourced materials, also called biological materials, which include for example polymers extracted from cells, food materials (see section 5.5 of this Roadmap), and active biological materials (see section 6.1). [2] Materials composed of chemically synthesized molecules identical, or similar to, macromolecules synthesized by living organisms. Examples include solid phase synthesis of nucleic acids or peptides, which can be building blocks for DNA or protein-based materials (see section 6.2 of this Roadmap). [3] Materials (biosourced or synthetic) used for a biological and/or medical application, such as scaffolds for mineralization for bone regeneration (see section 6.4).

Biosourced soft materials have a long history, going back to adhesives based on birch bark tar in the Paleolithic, papyrus in ancient Egypt, and the age-old use of leather and wool for clothing. Biological manufacturing is not limited to multicellular organisms like plants and animals. Bacterially produced polymers such as gellan gum are for instance popular rheology modifiers in the food industry. More recent “cell factories” include mammalian cells for biomedical materials, and microorganisms (yeast, bacteria, microalgae) for bioplastics, biominerals, and hierarchically structured composites [1, 2] (Figure 1). Recent developments in synthetic biology make it even possible to spatiotemporally control cells to produce materials on demand [3].

Biosourced materials have many advantages. A first obvious advantage is their high abundance. Cellulose for example constitutes >40% of the dry mass of plants. Second, they are inherently eco-friendly since biological organisms perform “green” chemistry. Third, their inherent biocompatibility makes them suitable for food and biomedical applications. Fourth, materials produced by living organisms often have superior material properties due to hierarchical structuring across multiple length scales [4]. Animal skins are for instance compliant yet resilient by virtue of a strain-stiffening fibrous collagen matrix combined with stretchy elastin, while bone and nacre are hard yet tough due to a layered structure with alternating soft protein polymer and rigid biomineral phases. Finally, many biosourced materials display additional features such as special optical or magnetic properties.

Engineered living materials, i.e., biosourced materials containing living cells, promise many additional advantages. Embedded motile microbes or contractile animal cells can serve as actuators that actively deform the material [5]. Cells can also render the material properties responsive to chemical cues or physical inputs, either naturally or through genetically engineered novel functionalities [3, 6]. Extracellular matrix synthesis by animal cells is for instance naturally mechanosensitive, as illustrated by the ability of bone to adapt its structure to mechanical load. Cells can endow materials with many additional life-like properties such as autonomous self-repair and programmed responses based on genetic circuits [3]. Such smart materials can open up many new applications, for instance as soft robots, intelligent food packages, recyclable bricks, and wearable bioelectronics[6].

## Current and Future Challenges

1. Most commercial biosourced materials such as plant-based cellulose or animal-based collagen are obtained by processing and/or extraction from natural sources. Although the materials are themselves eco-friendly, their insolubility often requires processing steps that involve harsh
chemicals and/or high energy input. Also, it can be challenging to ensure product purity, safety, and reproducibility; and especially animal-derived products raise ethical and cultural concerns.

2. These challenges can be overcome by sourcing biomaterials from wild type or engineered cell factories. Microbial fermentation is increasingly used for producing nucleic acids (DNA, RNA), proteins, polysaccharides, and biominerals [1]. However, it is not always straightforward to engineer novel pathways for materials production without compromising cell growth or viability [1]. Protein production yields are often lower than in endogenous organism and host organisms often lack the biosynthetic machinery needed to produce the correct posttranslational modifications. Polysaccharides can generally be produced in higher yields than proteins, but it is more challenging to engineer their production because our knowledge of their synthesis and processing pathways is incomplete [7]. Biomineral production is even more complex because it is environmentally sensitive.

3. Nucleic-acid-based materials are attractive because their properties can be programmed with rational computer-aided design of base-pairing DNA sequences. However, DNA-based materials are limited in their chemistry (4 DNA bases) and lack mechanical strength. Protein-based materials offer more versatile chemistry (20 amino acids, expandable with non-natural amino acids and posttranslational modifications) and higher mechanical strength. However, rational protein design is still hampered by our limited understanding of protein folding and self-assembly (see Section 6.2 of this Roadmap). Polysaccharides likewise offer versatility, but their physico-chemical properties are relatively poorly understood.

4. Current engineered living materials are far away from mimicking the complex shapes and internal structures of natural materials, although recent efforts to grow co-cultures of cells engineered to produce materials upon specific local triggers promise enhanced control [3]. Maintaining material production and performance over periods longer than a few days can be problematic due to nutrient and oxygen requirements and/or size constraints from cell growth. Some of these issues are also relevant in the field of tissue engineering [2].

<figure><img src="image_52.png" /><figcaption>**Figure 1** Schematic overview of the main biosourced cell and material categories.</figcaption></figure>

**Advances in Science and Technology to Meet Challenges**

1. A promising route towards more eco-friendly material extraction from natural biological sources is **enzyme-mediated extraction** [8]. Many natural enzymes exist that are selective for particular
biomacromolecules since living organisms depend on enzymes to dynamically sculpt their structure. Enzymatic processing proceeds under ambient conditions with low energy consumption, provides spatiotemporal control, and enzymes can add additional functionalities to the material [8]. Fundamental studies of the structure-degradation relationship for different materials are needed.

2. Production yields of cell factories can be enhanced by using synthetic biology tools to equip host cells with biosynthetic and secretory machineries from other organisms [1]. Directed evolution could further improve the performance of cell factories.

3. Achieving programmable material properties requires deep knowledge of the structure-property relation of biomolecules. Recent advances in artificial intelligence promise predictable protein folding from sequence. Advances in systematic coarse-graining computational approaches open the possibility to predict material properties from the underlying molecular properties, especially when combined with multiscale experiments exploiting new nanoscopic biophysical tools such as optical tweezers, atomic force microscopy, and molecular tension sensors [9]. Some of the new scattering and microscopy techniques developed for traditional soft matter systems would also be useful for studying biosourced materials (see sections 2.1 and 2.3 of this Roadmap).

4. New top-down fabrication processes such as molding, electrospinning, and 3D printing (Figure 2) provide a short-term strategy to achieve user-defined shape control of engineered living materials [10]. However, the holy grail in the field is autonomous cell-driven material assembly [3]. Synthetic biology makes it possible to achieve on-demand material synthesis by placing the corresponding gene(s) under the control of an inducible promotor and to pattern materials by fusing proteins with self-recognition motifs or by programming cell-material adhesion. Larger-scale patterning can be achieved by engineering cells that phase separate based on differential adhesion strengths or by using chemotactic responses of motile cells or stochastic reaction-diffusion mechanisms. Long-term performance stability of living materials will require a fundamental understanding of the behavior of cells confined within polymeric matrices.

<figure><img src="image_53.png" /><figcaption>**Figure 2** Examples of bioprinted engineered living materials composed of (A) microalgae or (B) bacteria, which demonstrate the possibility of controlling the 3D structure, cellular and molecular composition, and the functional properties of the printed living material. (A) Microalgae bioprints can survive several days off water and nutrients, and cells can be reused and grown to print additional living materials. (B) Emergent resistance of 7 day-old 3D-printed *E.coli* biofilms exposed to 70% ethanol during 10 min. The viability of bioprinted *E.coli* not producing (grey) and producing (orange) curli protein fibers is measured as colony-forming units (CFU) per mL. Parts of this figure are taken from Refs. [10, 11].</figcaption></figure>

**Concluding Remarks** Biosourced materials have a bright future because they provide a sustainable solution for many urgent societal challenges that demand safe and environmentally friendly materials. New engineered living materials further promise revolutionary new applications, such as self-repairing
construction materials or biologically active materials for personalized medicine. We expect many exciting future advances from the convergence of metabolic engineering and synthetic biology with (soft) materials science [3]. There are several important issues to address. The nomenclature in this new field is still being developed and would benefit from standardization. Recent papers have proposed a taxonomy for engineered living materials [12, 13]. Also, it is important to find ways to scale up fabrication methods to allow real-world applications. Finally, it is important to engineer the materials such that they are safe by design. To prevent release of the embedded cells to the environment, either the material can be designed to retain the cells by entrapment or adhesion, or the cells can be designed so they survive only inside the material.

**Acknowledgements** This publication is part of the project *How cytoskeletal teamwork makes cells strong* (with project number VI.C.182.004 of the NWO Talent Programme which is financed by the Dutch Research Council (NWO)), and the project *Light-responsive microalgal living materials* (ERC starting grant No. 101042612). Images are created with *BioRender.com*.

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# Section 6.4 – Reinforced Hydrogels via Mineralization: Next Generation Bioscaffolds

Rosa M. Espinosa-Marzal, Joaquin Yus and Jiheon Kwon

University of Illinois at Urbana-Champaign, Illinois, USA

**Status** Reinforced hydrogels have the potential to address the growing need for biomaterials that can support the growth and differentiation of stem cells, deliver drugs to specific tissues or organs, promote tissue regeneration (1) and ultimately lead to improved clinical outcomes in regenerative medicine.(2) Many organisms direct mechanisms referred to as biomineralization to produce a wide array of organic-inorganic composites with a broad spectrum of mechanical properties, spanning from hard and fracture resistant to soft and tough (3, 4). This remarkable variety is mediated by cell-coordinated material assembly processes that govern the mineralization of (nano)structured macromolecular scaffolds. Organic scaffoldings may reduce the energetic barrier for nucleation, locally raise the supersaturation of the solution, and/or direct the growth and assembly of the crystals within the organic matrix(5). One outstanding example is bone, which exhibits high levels of precision yielding multi-scale hierarchical gradients between soft organic matter and mineral phase, i.e., collagen and calcium phosphate. Bone growth appears to be templated by the collagen matrix, which leads to uniaxially oriented hydroxyapatite (HAP) nanocrystals parallel to the collagen fibers. The use of amorphous precursor phases is a widespread strategy in biomineralization(6, 7). In bone mineralization, it is proposed amorphous calcium phosphate (ACP) nanoparticles form first and gradually transform to hydroxyapatite (HAP) (8, 9). There is also recent evidence that amorphous calcium carbonate (ACC) may play a key role in mineralizing vertebrate tissues like bone (10).

Abiotic mineralization pathways have been intensively investigated aiming to gain control over material properties of nanostructured composites for biomedical applications. Hydrogels are often used to template mineralization. They possess a three-dimensional porous network, and therefore, microcavities, where the liquid composition can be controlled through both the polymer functional groups and the mesh size (11, 12). However, the relation between mineralization pathway, the microstructure of the mineralized hydrogels and their enhanced mechanical properties has only received attention recently. The projection is promising; a substantial reinforcement was achieved through the enzymatic precipitation of amorphous nanoparticles of calcium phosphate distributed homogenously within a hydrogel (13). The enhanced hydrogel toughness surpassed what can be achieved with (non-interacting) nanoparticles (fillers). It also proved the efficient coupling between mineral growth dynamics and polymer network mechanics (14). Moreover, this system promoted cell attachment, proliferation and differentiation and provided sites for bone osteogenesis (15, 16). Despite these advances, the fundamental principles dictating the relation between mineralization pathways in hydrogels, their microstructure, and mechanical reinforcement, and how it influences bone regeneration, remain greatly unexplored.

**Current and Future Challenges** Unrevealing this relation requires solving several challenges. The influence of the polymer concentration on nucleation is critical for the microstructure of the mineralized organic scaffold. This is intricate. On the one hand, it can lead to “shadow” effects –as the polymer may hinder collisions between clusters and prevent them from reaching a critical size. On the other one, it can provide low-surface energy nucleation sites that favour nucleation. In the context of non-classical nucleation, more studies are needed to fully understand how the assembly of minerals in the organic scaffold is affected or directed by the presence of highly ionic polymer-
induced liquid precursors. Strong interactions between the mineral and the organic phase are believed to, e.g., disturb the typical spherulitic structure of HAP in abiotic studies. However, there is still a lack of knowledge of the underlying principles.

The overall toughening of hydrogel-like scaffolds goes beyond the effects of nucleation. A crystal surrounded by a supersaturated solution exerts a thermodynamic pressure against the polymer network. Crystal growth against the polymer happens only if the disjoining pressure between mineral and crystal is smaller than this thermodynamic limit. If the polymer resists, the mineral traps the polymer during its growth (11). This disjoining pressure results from Derjaguin-Landau-Verwey-Overbeek (DLVO) and non-DLVO forces, which are greatly unknown for mineral-organic systems and difficult to measure or predict (Figure 1)(17). Polymer and solution compositions alter the disjoining pressure and could be a control mechanism for the polymer-mineral interaction, including the polymer inclusion. How amorphous precursors might affect such interaction remains also to be examined. Perhaps nanodroplets coalescing along the polymer network form a wetting film first. This film could solidify into an amorphous phase and eventually crystallize, while retaining the shape delineated by the wetting film along the polymer.

Predicting strengthening of hydrogels via mineralization poses additional challenges. Rubber elasticity models using adequate network theories could help test various hypotheses, including the often ignored but expected interactions between polymer and mineral. Polymer/mineral affinity could justify a more prominent reinforcement than simple fillers.

**Advances in Science and Technology to Meet Challenges**

Traditional hydrogels are often too weak to withstand the mechanical requirements of tissue engineering applications, which calls for new strategies for targeted reinforcement. Hydrogel mineralization can both improve mechanical support and maintain biocompatibility, and thereby promote the growth and differentiation of stem cells and tissue regeneration. Progress in this field requires the convergence of several disciplines, including (bio)mineralization, colloidal science, polymer physics, rheology, and cell biology. Figure 2 showcases recent developments in understanding the fundamental principles that guide the mineralization pathways in hydrogels. This work focused on calcium phosphate mineralization in agarose hydrogels via ACC precursors (18). It is important to realize that methods that rely on the drying of the composites need to be considered cautiously. Drying can alter both the mineral phase and the composite microstructure. Hence, *in situ* infrared or Raman spectroscopy are ideal to examine the (co)existence of amorphous and crystalline minerals in hydrated hydrogels. They can also be useful to reveal the chemical bonds between mineral and polymer. Environmental scanning electron microscopy and dynamic light scattering can reveal how nanoparticles grow, dissolve, and reaggregate inside hydrogels. This previous work (18) showed ACP nanoparticles were larger, and the number density smaller in the absence of ACC, reflecting that ACC promoted ACP nucleation. Small-angle X-ray scattering could reveal information about interparticle interactions. The observed particle aggregation can be compared with the stability ratio based on DLVO theory to understand the mechanisms that favour or hinder aggregation(19). However, accounting for non-DLVO phenomena, like charge reversal and adhesion from ion-ion correlations, will be more challenging. In colloidal science, a common strategy to elucidate colloidal stability is to vary ionic strength, ion valency and temperature, which could be applied in future research.

Figure 2c shows that the presence of ACC also changed the morphology of HAP precipitated in agarose hydrogels and strengthened the gel. Classical models to predict the strengthening of gels by fillers predict much smaller elastic moduli than the actual values (Fig. 2f), supporting the achieved
strong mineral-polymer interaction. It is possible that the smaller size of the ACP nanoparticles in the presence of ACC slowed down HAP crystallization, enhanced the liquid-like behaviour of ACP, and its wetting properties. All this could justify the different morphology of HAP and the strong interaction with the polymer. Nevertheless, studies that relate mineralization pathways with microstructure and mechanical properties of hydrogels are still needed to prove these hypotheses.

**Concluding Remarks** While hydrogels functionality and biodegradability make them suitable as scaffolds for tissue engineering, the most prominent challenges are still their inadequate mechanical strength and insufficient bioactivity. Achieving control over the strengthening of hydrogel-like scaffolds via mineralization relies on elucidating the relation between polymer properties, mineralization pathways and microstructure, the effect of solution chemistry, and the strengthening mechanisms. A suite of characterization techniques is available to examine these relations and experiments can be designed to explore specific mechanisms. Nevertheless, the parameter space is wide as not only mineral and solution composition but also polymer properties like polymer functionality, crosslinking type and self-assembly could significantly alter the mineralization pathway and the strengthening mechanisms. The delineated roadmap will be valuable to advance the knowledge and, among others, the use of mineralized hydrogels as bioscaffolds for bone regeneration. Further research will be needed to relate the principles underlying mineralization of hydrogels to their bioactivity.

**Acknowledgements** This material is based upon work supported by the National Science Foundation under Grant Nos. CMMI-2035122, CMMI-1435920, and Convergence RAISE program IOS-1848671.
Figures

<figure><img src="image_54.png" /><figcaption>**Figure 1** Measurement of mineral-polymer interactions. a) Light microscopy images of a calcite crystal glued to the AFM cantilever (red dashed square highlights the crystal) and b) SEM image of the calcite crystal glued to the cantilever. (c–d) Force–indentation curves upon extension (filled markers) and retraction (empty markers) at constant velocity of 20 nm/s measured with a calcite crystal glued to the AFM cantilever on (c) agarose hydrogels (1 wt %) and (d) a calcite crystal fixed to the AFM stage, both in a saturated calcium carbonate solution. The low adhesion energy between agarose and calcite (c) suggests the action of a disjoining force between them, which is also expected to exist during the growth of calcite within the hydrogel network. In comparison, this interaction becomes strongly attractive between two calcite crystals (d). Adapted with permission from Lopez-Berganza JA, Chen S, Espinosa-Marzal RM. Tailoring Calcite Growth through an Amorphous Precursor in a Hydrogel Environment. Crystal Growth & Design. 2019;19(6):3192-205. Copyright 2019 American Chemical Society.</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>**Figure 2** HAP mineralization in agarose hydrogels in the presence of ACC precursors. a-b) SEM images of coexisting ACP and ACC after 30 and 60 min mineralization, respectively. c-d) SEM images of the mineralized hydrogels; the morphology of apatite and its affinity to the polymer are altered upon mineralization via the ACC precursor. e) ACP nanoparticles growth, dissolution and aggregation in hydrogels, as measured by Dynamic Light Scattering at various equimolar concentrations of CaCl₂ and NaCO₃, Ca/P-ratio=1. f) Storage (G'')</figcaption></figure>
and loss modulus (G'') as a function of HAP volume fraction. e-f) correspond to HAP mineralization via ACC precursor. ACC prolonged the stability of ACP and retarded the crystallization into HAP. Spherulitic HAP-clusters formed in the absence of ACC, while ACC led to the formation of HAP platelets deposited along the agarose network, reflecting the stronger interaction between agarose and HAP. a-e) Adapted with permission from J Lopez-Berganza JA, Fu B, Lee C-WJ, Rogers SA, Espinosa-Marzal RM. Mediating the Enhanced Interaction Between Hydroxyapatite and Agarose through Amorphous Calcium Carbonate. Crystal Growth & Design. 2020;20(10):6917-29. Copyright 2020 American Chemical Society.

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