Hierarchically Engineered Artificial Lamellar Bone with High Strength and Toughness

Complex hierarchical architectures are ubiquitous in natural hard tissues, which comprise an elaborate assembly of hard and soft phases spanning from the nanoscale to the macroscale. The elegant architectures grant unique performance in terms of strength and toughness, but the biomimetic fabrication of synthetic materials with highly consistent structural and mechanical characteristics with natural counterparts remains a great challenge. Here, a centimeter‐size artificial lamellar bone is successfully fabricated for the first time via a well‐orchestrated “multiscale cascade regulation” strategy combining multiple techniques of molecular self‐assembly, electrospinning, and pressure‐driven fusion from molecular to macroscopic levels. The bulk artificial lamellar bone that is composed of hierarchically assembled mineralized collagen fibrils with a waiver of any synthetic polymer highly resembles the chemical composition, multiscale structural organization, and rotated plywood‐like structure of natural lamellae, thus achieving a good combination of lightweight and high‐stiffness (Ey ≈ 15.2 GPa), ‐strength (σf ≈ 118.4 MPa), and ‐toughness (KJC ≈ 9.3 MPa m1/2). This multiscale cascade regulation strategy can break through the limitations of a single technique and enable the construction of elaborate composite materials with multiscale step‐by‐step regulations of hierarchically structural organizations for unique mechanical properties.

DOI: 10.1002/sstr.202200256 Complex hierarchical architectures are ubiquitous in natural hard tissues, which comprise an elaborate assembly of hard and soft phases spanning from the nanoscale to the macroscale. The elegant architectures grant unique performance in terms of strength and toughness, but the biomimetic fabrication of synthetic materials with highly consistent structural and mechanical characteristics with natural counterparts remains a great challenge. Here, a centimetersize artificial lamellar bone is successfully fabricated for the first time via a wellorchestrated "multiscale cascade regulation" strategy combining multiple techniques of molecular self-assembly, electrospinning, and pressure-driven fusion from molecular to macroscopic levels. The bulk artificial lamellar bone that is composed of hierarchically assembled mineralized collagen fibrils with a waiver of any synthetic polymer highly resembles the chemical composition, multiscale structural organization, and rotated plywood-like structure of natural lamellae, thus achieving a good combination of lightweight and high-stiffness (E y % 15.2 GPa), -strength (σ f % 118.4 MPa), and -toughness (K JC % 9.3 MPa m 1/2 ). This multiscale cascade regulation strategy can break through the limitations of a single technique and enable the construction of elaborate composite materials with multiscale step-by-step regulations of hierarchically structural organizations for unique mechanical properties.
structural unit of lamellar bone contains five successive layers of parallel mineralized collagen fibrils with their orientation increasing %30°to form a rotated plywood-like structure. [20] It is considered that anisotropic and compact lamellae are critical for the excellent mechanical properties of natural bone. [21] To mimic the plywood-like structure of natural lamellar bone, a well-orchestrated facile "multiscale cascade regulation" strategy was developed in our group ( Figure 1). First, a collagen-induced biomineralization process is precisely controlled to form mineralized collagen (MC) microfibrils at the nanoscale level via molecular self-assembly. Then the MC microfibrils are added into a collagen solution for electrospinning to further make MC fibrils that are organized into random or aligned arrays after collection. The electrospun MC fibril layers that are designated as the sublayer analogs of lamellar bone units stack together with successive rotations to form plywood-like structures at a higher level of organization. Finally, a highly integrated bulk laminate is obtained via a pressure-driven fusion process at room temperature, which is defined as artificial lamellar bone (ALB).

Hierarchical Structure of ALB
The synthetic ALB shares a similar structural organization with a natural lamellar bone, which highly recapitulates the hierarchical arrangement of MC microfibrils from the nanoscale to the macroscale (Figure 2A). At the nanoscale level, biomimetic mineralized collagen microfibrils with a diameter of about 8 nm gradually precipitate in the biomimetic mineralization process, during which amorphous calcium phosphate (ACP) precursors first nucleate on the surfaces of the preassembled collagen microfibrils at the early stage of mineralization (Figure S1B, Supporting Information). As the mineralization progresses over time, the orderliness of the MC microfibrils increases gradually accompanied by the transformation of ACPs to crystalline apatite ( Figure S1C,D, Supporting Information). The in situ coassembling process of nHAp and collagen microfibrils was confirmed by the evidence of high-magnification transmission electron microscopy (TEM) morphologies and fast Fourier transform (FFT) patterns ( Figure S1E-G, Supporting Information). The selected area electron diffraction (SAED) patterns show the typical diffraction rings of nanocrystalline hydroxyapatite ascribed to crystallographic planes (002) and (211), which is confirmed by the crystal structure imaged by TEM ( Figure 2B). The electrospun MC fibrils with a uniform diameter of approximately one hundred nanometers imitate the naturally self-assembled mineralized collagen fibrils for the subsequent organization at higher levels ( Figure 2C and S2, Supporting Information). The MC microfibrils evenly distribute within the electrospun MC fibrils and align with their long axes along the fibrils under the action of the high electric field and the shear force on the pinhole tip during electrospinning. The SAED pattern exhibits polycrystalline rings of hydroxyapatite with a typical (002) preferential orientation of their crystallographic c-axis parallel to the longitudinal direction of collagen fibrils ( Figure 2C (insert)).
At the microscale level, the electrospun MC fibrils were collected into layers with aligned or random fibril arrays that are ubiquitous in natural bone tissues ( Figure S2, Supporting Information). The aligned MC fibril layers were stacked together with their orientations increasing by 30°to simulate a lamellar unit and form a centimeter-size compact bulk bone maintaining the initial fibril orientations and structural arrangements after pressure-driven fusion ( Figure 2E,F and S3, Supporting Information). In addition, the thin foils in parallel and perpendicular fiber directions of one sublayer obtained by the focused ion beam (FIB) were observed by scanning transmission electron microscopy (STEM), which showed that the MC microfibrils and  www.advancedsciencenews.com www.small-structures.com fibrils maintained the orientation characteristics ( Figure 2D). The final bulk ALB has a quite similar appearance to natural cortical bone, and the fracture surface reveals a compact and laminated lamellae-like microstructure ( Figure 2F). The density (ρ) of the bulk ALB could reach 1.485 g cm À3 , which is slightly lower than that of natural cortical bone because of the lower apatite content in ALB ( Figure S4, Supporting Information). Moreover, by adjusting the fibril orientations or the angles between the two adjacent fibril arrays, other biomimetic compact bone materials with different organizational patterns are easily obtained, including arrays of parallel fibrils (PFBs), random fibril woven structure (RFB), and orthogonal fibril plywood-like architecture (OFB) ( Figure S5, Supporting Information).

Mechanical Properties and Toughness Mechanism
It's known that the complex hierarchical architectures of natural materials contribute to their unique mechanical properties. [21][22][23] It is therefore remarkable to know if the mechanical properties of ALB are optimized by the biomimetic hierarchical structures.
The microscopic mechanical properties of the ALB constructed under different pressures (P) were studied by nanoindentation. The Young's modulus (E y ) and surface nano-hardness (H) was calculated using the load-displacement curves ( Figure S6, Supporting Information). As P increased to 1000 MPa, the E y and H of the resulting ALB increased gradually and reached 15.2 AE 0.2 GPa and 403.0 AE 11.0 MPa, respectively ( Figure 3A). The SEM morphologies of the residual indent impression show a striking similarity with that of natural bovine cortical bone, exhibiting a typical profile of ductile materials and excellent crack resistance performance with no microcracks induced by the indents ( Figure 3B). Furthermore, the macroscopic mechanical properties of the ALBs are improved with the increase of the briquetting pressure, gradually approaching the level of natural bone ( Figure 3C,D). The ALB prepared under a pressure greater than 1000 MPa exhibits excellent mechanical properties with an ultimate tensile strength of 72.0 AE 1.5 MPa by the tensile test ( Figure S7 and S8, Supporting Information). In addition, similar to the anisotropic mechanics of natural bone, the ALBs along different directions exhibited anisotropic flexural strength (σ f ) and modulus (E f ) of 118.4 AE 5.6 MPa and 95.6 AE 7.2 GPa in antiplane direction (with the loading direction perpendicular to the laminated layers) and 67.9 AE 2.6 MPa and 66.9 AE 2.3 GPa in the in-plane direction (with the loading direction parallel to the laminated layers), respectively ( Figure 3E and S9, Supporting Information). Furthermore, we fabricated three artificial bone materials, ALB, PFB, and RFB with rotated, parallel, and random fiber orientations, respectively, and tested their mechanical properties by single-edge notched three-point bending (SENB) tests with loading along the in-plane or antiplane direction ( Figure 3E (right) and S10, Supporting Information). Crack-resistance curves (R-curves) of these artificial compact bones show an increasing tendency, indicating their good crack-resistance to fracture ( Figure 3F). The ALB architecture shows the highest fracture toughness (K JC % 9.3 MPa m 1/2 ) in the antiplane direction, and the ALB, PFB, and RFB have modest fracture toughness in the in-plane direction, which implies that the overall fracture toughness can be improved by the lamellar structure ( Figure 3G and S11, Supporting Information). In comparison to a range of typical biological materials and synthetic engineering materials, the mechanical properties of ALB material perform outstandingly, comparable to the natural cortical bone in stiffness and hardness but superior in specific strength and toughness ( Figure 3G,H). The statistical results of the typical mechanical properties are listed in Table S1-S3, Supporting Information.
The outstanding mechanical performance of ALB is conferred by its composite components and hierarchical architecture from nanometer to millimeter through associated intrinsic and extrinsic toughening mechanisms at multiple length scales. As the basic building block of ALB, MC fibril is a typical nanocomposite of nanoplatelets of calcium phosphate minerals and collagen molecules, which is confirmed by the 3D element distributions of Ca-O (representative of inorganic phase) and C-OH (representative of organic phase) by atom probe tomography (APT) ( Figure S12, Supporting Information). The well-organized MC fibrils contribute to the general mechanical performance of ALB at the nanoscale, which is ascribed to the high stiffness and strength of these uniformly distributed mineral nanoplatelets and the intrinsic toughening mechanisms of fibril rotation, sliding, pull-out, bridging, interfibrillar debonding, and interfacial friction and sliding between the minerals and collagen molecules. [24] Moreover, the unique plywood-like structure of ALB with rotated MC nanofibril orientations and laminated features optimizes the toughness, [21,23,25] leading to energy dissipation occurring at the microscale level. To verify the effect of MC fibril orientations and hierarchical structure on the strength and toughness of the artificial compact bones. The in situ SEM tensile tests of RFB and PFB architectures that visualize crack initiation and propagation processes under stress confirm the toughening effect of fibril orientations (Figure 4 and S13, Supporting Information). The aligned fibril structure contributes to fibril bridging, crack branching, and crack deflection behaviors in the crack initiation and propagation path. However, the crack in RFB architecture shows a straight propagation path along the loading direction, which suggests that the interwoven structure of random MC fibrils has no obvious contribution to external toughening. The blunt U-type crack tip at the crack initiation stage should mainly be ascribed to internal toughening.
The crack deflection/twist, crack branching, microcracks, and fibril bridging also occur during crack propagation in ALB owing to the external toughening mechanisms and lead to its excellent toughness without sacrificing strength ( Figure 5A,B). More than that, the quick-rising R-curve of ALB (antiplane) indicates its good fracture resistance ability that originates from the interlamellar crack deflection and debonding. The corresponding fracture surface morphologies of ALB in the antiplane direction illustrate that the lamellated structure contributes to crack redirection/ twist and crack-induced interlamellar debonding and sliding at the interfaces for energy dissipation ( Figure 5D and S14, Supporting Information). Besides, the SEM images of the fracture surface show a longer stable crack growth distance in ALB (in-plane) than that in RFB (in-plane), conforming to the higher toughening effect of the rotated fibril orientations ( Figure 5E, S15 and S16, Supporting Information).

Pressure-Driven Fusion Occurs in ALB Bulk Formation
The external high pressure is key to drive the fusion and densification of the stack of electrospun MC fibril layers into a bulk laminated material. At P < 250 MPa, insufficient interlaminar integration occurs, and layers could be separated by a simple peel-off process, although there is an obvious fiber bridging occurring on the fracture surface of the cross-section ( Figure S17, Supporting Information). As the external P increases gradually to higher than 350 MPa, the MC fibril layers coalesce completely, forming a compact lamellar-like material that cannot be stratified easily by external mechanical force ( Figure S3 and S18, Supporting Information). We attributed the increasing modulus and hardness by nanoindentation tests to the fusion and aggregation of MC microfibril ( Figure S19, Supporting Information).
In natural bone tissue, it is demonstrated the inorganic minerals that are initially deposited in the hole zones of assembled collagen fibrils grow across collagen fibrils and fuse in coplanar alignment to form larger mineral platelets. [19] Inspired by this phenomenon, we speculate that the integration of the bulk ALB www.advancedsciencenews.com www.small-structures.com is probably driven by the fusion of apatite crystals under P.
To further explore the role of inorganic minerals on the pressure-driven fusion, we prepared a series of MC fibril layers containing different contents of apatite minerals, which were found to be unable to sufficiently integrate into a good bulk ALB material with a mineral content of less than about 20% by weight. In addition, the electrospun pure collagen fibrils or with commercially high-crystalline HA nanoparticles will not fuse to a bulk material under external P either ( Figure S20, Supporting Information), indicating that the amorphous/low-crystalline minerals deposited under the regulation of collagen molecules during the in vitro biomimetic biomineralization process may be particularly important for P-induced interlamellar fusion. [26] We then performed Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectra to analyze the changes in the inorganic phases in response to external pressures ( Figure 6A-C).
In the FTIR spectra, the ratio of the integrated areas of phosphate (900-1200 cm À1 ) and amide I (1650 cm À1 ) that can provide a measure for the total amount of apatite minerals shows an obvious rising tendency as the pressure increases, suggesting an increase in the apatite content ( Figure 6B and S21, Supporting Information). The raw peak of phosphate corresponds to the phosphate ν 1 , ν 3 band that consists of a dozen underlying peaks, each of which represents a specific phosphate environment ( Figure S22, Supporting Information). [27] It is noted that there is a clear increase in the relative areas of nonstoichiometric phosphate structure peaks, including 1054, 1074, 1096, and 1113 cm À1 ( Figure S23, Supporting Information), which implies that the increased amount of minerals is probably attributed to the crystallization of amorphous calcium phosphate particles into low  www.advancedsciencenews.com www.small-structures.com crystalline apatite. Besides, there is a dramatic drop in FTIR absorption ( Figure S21, Supporting Information) for the ALB samples prepared at pressures larger than 250 MPa, which further confirms the compact bulk formation with the improved internal continuity and elimination of pores and voids under high P. The optical photograph of a thin sample of ALB even reveals a transparent appearance ( Figure 6D and S24, Supporting Information). More than that, XRD spectra show a steep ascent in the intensity of the (211) peak with pressures larger than 250 MPa, suggesting that the mineral crystallinity increases and the crystalline fusion in the crystallographic axis (211) reflections may occur ( Figure 6C). We, therefore, speculated that the external pressure could also promote the phase transformation of the internal apatites from a low crystalline state to a highly crystalline state accompanied by their coalesce. The electrospinning process made the preferentially deposited HAp nanocrystallites orient inside the aligned collagen fibrils with the neighboring crystals in a nearly parallel arrangement like a "deck of cards", thus providing the potential opportunity for them to fuse and grow ( Figure 2D). External high pressure may drive these crystals to rotate with an improved preferential orientation that is more conducive to crystal fusion and growth. The grain size distribution determined by XRD displays a significant increase with increasing external P ( Figure S25, Supporting Information). Furthermore, the micro-computed tomography (μ-CT) images directly visualize the mineral aggregations, confirming the increased particle size ( Figure 6E and S26, Supporting Information). In fact, a similar pressure-driven fusion of amorphous particles into integrated monoliths has previously been reported, which proves that structurally bound water promotes mass transportation for particle fusion through dynamic water channels under pressure. [28] A similar process likely exists in our work. The tiny apatite minerals formed through the in vitro biomineralization process have low crystallinity/maturation and bound water ( Figure S4, Supporting Information), which assists the diffusion of inorganic ions in water channels as compared to the synthesized HAp at high temperature with sintering. This also explains why sintered HAp with high crystallinity cannot drive the fusion of the electrospun collagen/HAp composite. In brief, our results above imply that the fusion and transformation of inorganic phases within the MC fibrils contribute to the formation of the compact ALB under pressure.

Bioactive ALB and in vitro Biocompatibility
To further endue ALB with good bioactivities, bioactive chemicals such as growth factors, drugs, and inorganic ions can be easily added during the biomimetic mineralization process to make bioactive ALBs because of the mild reaction conditions and preparation processes. [29][30][31] For example, we successfully synthesized a variety of ion-doped MC fibrils with the substitution of Sr, Si, or Mg, in apatite to mimic the inorganic minerals in the natural bone that are not pure HAp crystals, but ion-substituted apatites instead. As shown in Figure 7, the bone marrow-derived mesenchymal stem cells (BMSCs) showed very good attachment, spreading, and proliferation on the three samples, collagen membrane, ALB, and Sr-doped ALB. And the doping of Sr in hydroxyapatite did not affect the biocompatibility of ALB.  Bioactive ALB can be considered a "real bone" substitute for a wide range of clinical applications in oral, neurosurgery, and orthopedics owing to its biomimetic chemical composition, hierarchical structure, and unique mechanical performance and bioactivity. And more advanced biomimetic architectures such as an osteon or a long bone may also probably be constructed using this strategy. Furthermore, this facile "mineralization-assembly-compression" cascade processes can be easily scaled up in production, possessing great potential for product transformation.

Conclusion
In summary, we successfully engineered a centimeter-sized artificial lamellar bone via a well-orchestrated "multiscale cascade regulation" strategy combining multiple techniques of molecular self-assembly of collagen and calcium phosphate nanocrystals, electrospinning, and pressure-driven fusion process. ALB contains highly consistent organic and inorganic components with natural bone tissue with a waiver of any synthetic polymer. Specifically, we replicate the multiscale structural organization with a broad spectrum of hierarchies spanning from the nanoscale to the macroscale, which defeats the conflict of strength and toughness and achieve a good combination of ultra-lightweight and high-stiffness, -strength, and -toughness. In addition, given the importance of amorphous/low-crystalline minerals for bulk material formation under external pressure, we speculate that our strategy can provide a facile method to construct various compact organic/inorganic composite materials with unique mechanical performances.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.