Bioprocessing-Inspired Programmed Collagen Intrafibrillar Mineralization Controlled by Ligand Chain Length of Gold Nanoclusters

Wed, 03 Jun 2026 12:00:00 +0800

On June 3, 2026, the research team led by Researcher Zhaoyong Zou, Associate Researcher Luyao Yi, Professor Guanbin Gao, and Academician Zhengyi Fu from the State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, in collaboration with Professor Peter Fratzl from the Max Planck Institute of Colloids and Interfaces, Germany, published their findings entitled “Bioprocessing-Inspired Programmed Collagen Intrafibrillar Mineralization Controlled by Ligand Chain Length of Gold Nanoclusters” in Advanced Functional Materials.

1. Research Background

The biomineralization process of bone exemplifies nature’s intelligence in building materials, demonstrating unique design principles for creating hierarchically ordered, high-strength biocomposites. The precise integration of flexible collagen fibrils with rigid intrafibrillar nanoscale hydroxyapatite (HAP) crystals endows bone with remarkable mechanical resilience. Central to this precise spatial organization is the regulation by noncollagenous proteins (NCPs), which orchestrate a multi-step mineralization process: stabilizing transient amorphous calcium phosphate (ACP) liquid precursors, facilitating their infiltration into the intrafibrillar spaces of collagen, and directing their non-classical transformation into aligned HAP crystals along the collagen fibril axis.

Despite decades of research, the specific molecular features of NCPs that govern this intricate process—particularly the selection between intra- versus extrafibrillar mineralization pathways—remain incompletely resolved. Conventional polymeric NCPs analogues (such as polyacrylic acid PAA and polyaspartic acid PAsp), while effective in inducing liquid-liquid phase separation (LLPS) and promoting some intrafibrillar deposition, suffer from critical limitations: ill-defined structural conformations, inability to isolate the effects of specific molecular parameters, and poor electron contrast preventing direct visualization.

2. Research Content

Inspired by the function of NCPs during biomineralization, the team designed atomically precise, electron-dense ligand-engineered gold nanoclusters (AuNCs) as novel biomimetic NCPs analogues, achieving precise programming of collagen mineralization patterns through ligand chain length control.

Design and Synthesis of Gold Nanoclusters

The team synthesized two types of gold nanoclusters with different chain-length carboxyl ligands: AuNCs-MPA (3-mercaptopropionic acid, short-chain ligand) and AuNCs-MHA (6-mercaptohexanoic acid, long-chain ligand). Both contain carboxyl groups to mediate interactions with mineral ions, but different chain lengths result in divergent interfacial behaviors: MPA ligands impose short chain length-dependent steric constraints and effective rigidity similar to protein folding, while MHA ligands afford long chain length-dependent conformational flexibility with reduced steric hindrance. TEM, UV-Vis, FTIR, and XPS characterization confirmed the successful synthesis of both nanoclusters.

Molecular Dynamics Simulations Reveal Precursor Formation Mechanisms

Molecular dynamics (MD) simulations systematically compared the dynamic interactions between different NCPs analogues and mineral ions. Results showed that all systems containing NCPs analogues exhibited characteristic liquid-liquid phase separation (LLPS) behavior, forming chemically distinct solute-rich and solute-poor regions. In the solute-rich regions, large numbers of Ca²⁺ and HPO₄²⁻ ions were tightly bound to the NCPs analogues.

Interestingly, CaP clusters further induced rotation and folding of MHA ligands on the gold nanocluster surface, reducing spatial hindrance and exerting potential impacts on precursor stability. Simulations demonstrated that PAA and AuNCs-MPA systems exhibited greater stability, while pure ACP and AuNCs-MHA were more prone to rapid crystallization. Structural differences among NCPs analogues led to distinct assembly behaviors: significant variation in the relative distance between adjacent carboxyl ligands was observed between AuNCs-MPA and AuNCs-MHA systems.

Ligand Chain Length Determines Collagen Mineralization Patterns

Single-layer collagen fibril mineralization experiments revealed the decisive effect of ligand chain length on mineralization patterns:

AuNCs-MPA (short-chain) induces intrafibrillar mineralization: Successfully achieved intrafibrillar mineralization with characteristic D-banding structures—gap regions (~40.2 nm, high electron transmission) and overlap regions (~26.4 nm, low electron transmission). SAED showed characteristic HAP diffraction rings, and HRTEM confirmed ordered HAP crystal deposition along the c-axis. HAADF and EDS showed significant enrichment of AuNCs-MPA at the inner edges and overlap regions of collagen fibrils, with uniform Ca and P distribution. Electron density line profiles confirmed that the spatial distribution of AuNCs-MPA aligned with the periodic D-banding structure of collagen fibrils—high electron density in overlap regions and low density in gap regions.

AuNCs-MHA (long-chain) leads to extrafibrillar mineralization: Despite having similar carboxyl groups, only extrafibrillar mineralization was observed across a broad concentration range of 100–1000 μg/mL, failing to achieve intrafibrillar deposition.

Two Interconvertible ACP Liquid Precursor Types

TEM characterization revealed the simultaneous existence of two distinct types of ACP liquid precursors in the AuNCs-MPA system:

Particulate precursors: ~15 nm in size, with significantly low electron density, AuNCs-MPA (~1.3 nm) enveloped by Ca and P elements, highly dispersed
Aggregated precursors: Higher electron density, significantly increased Ca/Au and P/Au ratios, AuNCs-MPA aggregated into larger particles of ~4.0 nm

After 2 hours of reaction, aggregated precursors continued to accumulate mineral ions, displaying increased electron density and sharper phase boundaries. Interestingly, initial particulate precursors further assembled and densified into the aggregated state. In the AuNCs-MHA system, aggregated precursors exhibited a filamentous morphology, distinct from the spherical aggregates of AuNCs-MPA-ACP.

Crystallization Kinetics and Stability Control

Automatic potentiometric titration monitoring showed that pure ACP crystallized at approximately 6000 s, while MPA slightly delayed crystallization to approximately 12000 s. PAA and AuNCs-MPA effectively stabilized ACP liquid precursors (no significant pH or Ca²⁺ concentration decrease over 30000 s), consistent with their capacity to facilitate intrafibrillar mineralization. In contrast, AuNCs-MHA markedly accelerated the transformation of ACP to HAP (within only 580 s).

TEM demonstrated that crystallization in the AuNCs-MPA-ACP system initiated from small particulate precursors, which directly transformed into thin, plate-like crystals. FFT analysis identified characteristic diffraction spots corresponding to the (002) and (112) crystallographic planes of HAP. Smaller AuNCs-MPA (~1.2 nm) were uniformly incorporated within HAP crystals, while larger clusters (~2.3 nm) mainly accumulated at crystal edges. Aggregated precursors dissolved near HAP crystals and accumulated at the mineralization front, replenishing CaP clusters.

Direct Visualization of the Intrafibrillar Mineralization Process

Leveraging the high electron density of AuNCs-MPA, the team achieved—for the first time in experimental settings—direct visualization of NCPs analogue-mediated intrafibrillar mineralization dynamics at the nanoscale:

0–3 h: AuNCs-MPA predominantly localized at the junctions between overlap and gap regions of collagen fibrils (a-bands, ~9 nm wide), confirming specific electrostatic interactions with collagen fibrils
3 h: Particulate ACP liquid precursors adsorbed onto collagen fibril surfaces in an ordered manner, preferentially aligning along the a-bands and adjacent e-bands of the gap region, similar to early mineralization sites in the presence of PAsp
6 h: Initial precursors that infiltrated into gap regions began to transform into HAP crystals, resulting in lateral expansion of collagen fibrils
12–24 h: Crystal growth progressed along the longitudinal direction of collagen fibrils, with HAP crystals gradually filling fibrillar space both axially and radially
Late stage: After gap region mineralization, AuNCs-MPA were present not only in mineralized regions but also enriched in adjacent regions including inner surfaces and overlaps, subsequently infiltrating into overlap regions through precursor permeation and achieving densification

Ultrathin sections further revealed that aggregated precursors were exclusively observed outside collagen fibrils, dissociating and dispersing near mineralization fronts; particulate precursors were detected both within collagen fibrils and at mineralization fronts. This indicates that particulate precursors are the primary drivers of intrafibrillar mineralization, while the more stable aggregates serve as ion reservoirs for subsequent mineralization.

“Gap-to-Overlap” Sequential Mineralization Model

Based on these findings, the team proposed an NCPs-induced “gap-to-overlap” sequential collagen intrafibrillar mineralization model:

Infiltration into gap regions: Particulate ACP liquid precursors first infiltrate gap regions and transform into HAP crystals
Crystallization in gap regions: During crystallization, NCPs are partially incorporated into the crystal lattice and partially expelled
Infiltration into overlap regions: Expelled NCPs are confined to adjacent overlap regions and inner surfaces due to spatial constraints imposed by collagen fibrils, rather than being released externally
Crystallization in overlap regions: After gap regions are fully mineralized, overlap region mineralization initiates, achieving mineral filling of all spaces within collagen fibrils

This sequential mineralization process, coupled with the confined geometry of collagen fibrils, determines the specific enrichment of NCPs in the overlap zone.

3. Summary and Outlook

In this work, the team successfully developed ligand-engineered gold nanoclusters as a novel and potent class of NCPs analogues to elucidate the mechanism of collagen mineralization. Through short-chain (AuNCs-MPA) and long-chain (AuNCs-MHA) variants, they clearly demonstrated that ligand chain length-dependent conformational rigidity, mimicking the functional domains of NCPs, is a key determinant that directs mineralization toward either intrafibrillar or extrafibrillar pathways.

The study also unveiled the existence and distinct roles of two interconvertible ACP liquid precursors: dispersible nanoparticulate precursors as infiltration agents versus aggregated droplets as ion reservoirs. The proposed “gap-to-overlap” sequential mineralization model explains the specific spatial distribution of NCPs in mineralized collagen fibrils, providing new structural insights into the origin of bone’s exceptional toughness.

This study not only enhances our understanding of biological mineralization mechanisms but also establishes a robust bioprocessing-inspired fabrication platform for engineering high-performance biomimetic composites with precisely controlled hierarchical architectures and superior mechanical properties. Short-chain ligands are favorable for inducing mineralization within confined spaces, facilitating the in vitro fabrication of high-performance organic-inorganic composite materials and tissue repair scaffolds with hierarchical nanostructures mimicking natural bone and dentin. By contrast, long-chain ligands are more suitable for the fabrication of surface mineralized layers requiring rapid hardening, as well as resin and cement-based materials.

4. Publication Information

Luyao Yi, Xiaoyu Zhang, Jielong Gao, Jin Chen, Cui Huang, Taolei Sun, Guanbin Gao, Peter Fratzl, Zhengyi Fu, and Zhaoyong Zou, Bioprocessing-Inspired Programmed Collagen Intrafibrillar Mineralization Controlled by Ligand Chain Length of Gold Nanoclusters. Advanced Functional Materials 2026, e76228.

https://doi.org/10.1002/adfm.76228

Noncollagenous Protein Analogues | Bioprocess Inspired Fabrication