Bone is a remarkable tissue that exhibits an impressive capacity for self-healing. However, in cases of large defects, this innate regenerative ability is often insufficient. In such scenarios, the absence of a supportive template can significantly hinder the healing process, leading to complications in recovery and function. Traditional approaches to bone repair rely on autologous bone grafts, which are considered the gold standard for bone replacement. These grafts are highly effective due to their inherent biological properties, which include the presence of progenitor cells, osteoblasts, embedded in a mature extracellular matrix along with a rich supply of growth factors and an established vascular network.

Despite their advantages, autologous grafts have limitations, including donor site morbidity and the availability of sufficient bone for harvesting. This has driven research toward the development of alternative bone substitutes that can effectively support tissue regeneration. Ceramics, particularly bioceramics, are mineral-based materials widely used as scaffolds in bone tissue engineering. An important characteristic of such materials is the degree of crystallization, as materials like hydroxyapatite (HA), the most stable crystalline phase of calcium phosphate, shows optimal mechanical (stiffness) properties. Conversely, its resorption in the body is not optimal; thus, HA is often combined with β-tricalcium phosphate (β-TCP) to improve its solubility. When structured with a (bio)polymer, it can also direct bone neo-tissue organization through physical guidance, while the addition of growth factors supports and accelerates bone formation.

However, previous work has shown that the cooperative interactions between collagen and mineral significantly influence the final properties of bone tissue. In addition, the physical properties of the substrate are now considered crucial for driving the physiological cells phenotype expression (ref. 1). While the hierarchical organization of bone is known to play a critical role in its mechanical properties, the specific contribution of this pattern to its biological properties as a graft remains unknown. In other words, understanding how the bone microstructure interacts with cells and influence the healing process is still required to explain the performance of autologous bone grafts and build new promising models and biomaterials. Indeed, current biomaterials often lack a substantial biomimetism in terms of microstructure and composition (highly crystallized HA versus bioapatite and absence of collagen). They can be seen as predominant “stony” ceramic-implants that must be macroporous to allow host cells colonization, hence failing to reproduce bone hierarchical structure. Conversely, a promising pathway is the development of biomimetic materials that reproduce the complex biological environment of natural bone meaning that it not only replicates the chemical composition of bone—such as collagen and bioapatite—but also mimic its intricate three-dimensional hierarchical microstructure.

The Role of Microstructure in Bone Healing

Bone tissue is composed of an organic/inorganic hybrid matrix, primarily consisting of type I collagen fibrils and "poorly" crystalline apatite nanoparticles (i.e., an amorphous calcium phosphate coating is found around the carbonated apatite crystalline core, ref. 2). This unique composition provides bones with a combination of strength and flexibility (demineralized bone being a self-standing material, Figure 1). The organization of these components is hierarchical and can be categorized into various structural levels (nowadays described up to twelve, ref. 3), each contributing to the overall function of bone. For instance, both compact and trabecular bone share a fundamental twisted plywood pattern of mineralized collagen fibrils (level nine). This specific arrangement allows for high density and structural order, critical for the mechanical performance of bone and may also be crucial for driving the cellular behavior. 

Figure 1 (Supplementary Video 1)

Advancements in Biomimetic Materials

In light of this understanding, we have developed innovative cell-free biomaterials that not only replicate the composition of bone but also its three-dimensional microarchitecture. These advanced materials are engineered using state-of-the-art self-assembly techniques that allow for the precise arrangement of collagen and apatite, resulting in a structure that closely resembles natural bone, i.e., exhibiting the twisted plywood pattern of mineralized collagen fibrils (ref. 4).

One of the notable achievements in this field is the ability to replicate nine out of the twelve distinct levels of bone's hierarchical organization. This is a significant advancement compared to existing biomaterials on the market, which often only achieve one or two levels at most. By successfully reproducing this structural feature, the new biomimetic materials (with or without minerals) were tested in vivo (murine and ovine models, Figure 2a and b, respectively) to evaluate their efficacity as graft and further investigate the potential implications of physicochemical factors in bone repair and regeneration.

Figure 2

Comparative in vivo Studies and Results

To evaluate the efficacy of these biomimetic materials, extensive comparative studies were conducted against established autologous bone grafts and commercially available synthetic ceramics. A variety of methodologies, including histopathology, micro-computed tomography, wide-angle X-ray scattering, microindentation and electron microscopy, were employed to assess the biological, mechanical and structural performance of the materials. The results are very promising (Figure 3a). The biomimetic scaffolds demonstrate the capacity to significantly improve the quality of bone repair compared to ceramics, which often fail to resorb well and result in reconstructed bone without remodeling. The biomimetic materials not only promote cellular colonization but also allow for natural resorption and remodeling processes, leading to a more effective healing response.

Moreover, our investigations reveal that the density of collagen fibrils, the structural organization of the material, and the crystalline characteristics of the apatite phase are crucial for driving the bone repair mechanism. Specifically, the "most biomimetic structure" i.e., the twisted plywood pattern of the collagen matrix, combined with the poorly crystalline nature of the apatite, provides a favorable environment for cell attachment and proliferation, even without macroporosity, inducing angiogenesis and leading to mature bone formation (figure 3b). This unique interaction between the material and living cells underscores the importance of both the organic microstructure and the mineral composition in achieving successful bone regeneration and explains partly the autograft performances that are commonly discussed from a cellular and mechanical point of view.

Figure 3

Implications for Future Research

By demonstrating that the unique architectural design of the bone extracellular matrix can influence cellular behavior, we provide experimental evidence supporting a long-standing theory in tissue engineering (ref. 1). This opens up new ways of thinking the development of competitive biomaterials for bone repair and regeneration, as well as for establishing relevant models to improve our fundamental understanding of bone biomineralization.

References 

1. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).
2. Wang, Y. et al. Water-mediated structuring of bone apatite. Nat. Mater. 12, 1144–1153 (2013).
3. Reznikov, N., Bilton, M., Lari, L., Stevens, M. M. & Kröger, R. Fractal-like hierarchical organization of bone begins at the nanoscale. Science 360, eaa02189 (2018).
4. Wang, Y. et al. The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite. Nat. Mater. 11, 724–733 (2012).