Co-inspired hydroxyapatite-based scaffolds for vascularized bone regeneration

Abstract

Hydroxyapatite (HA) is the main inorganic component of human bone. Inspired by nacre and cortical bone, hydroxyapatite-based coil scaffolds were successfully prepared. The scaffolds presented “brick and mortar” multi-layered structure of nacre and multi-layered concentric circular structure of cortical bone. Because of bioactive components and hierarchical structure, the scaffolds possessed good compressive strength (≈95 MPa), flexural strength (≈161 MPa) and toughness (≈1.1 MJ/m³). In addition, they showed improved angiogenesis and osteogenesis in rat and rabbit critical sized bone defect models. By mimicking co-biological systems, this work provided a feasible strategy to optimize the properties of traditional tissue engineering biological materials for vascularized bone regeneration.

Introduction

After millions of years of natural evolution, nature organisms have obtained a combination of sophisticated structure and multi-functionality. Learning from the hierarchical structures of nature materials, researchers have produced high-performance materials ranging from nanometers to micrometers [1], [2], [3], [4], [5]. For instance, the 3D printed scaffolds with high porosity and good bioactivity inspired by lotus root [6,7], high-adhesion materials inspired by gecko and frog [8,9], bio-inspired superwettable surfaces or interface materials [10], [11], [12], [13] and other prominent biomimetic materials [14], [15], [16].

A classic problem of biological material design is that strength and toughness, the two key structural properties, tend to be exclusively reciprocal. Generally, materials with high toughness are invariably weak, whereas high-strength materials are frequently brittle [17]. Inspired by natural organisms, such as nacre, fish scales, teeth, and bone, structural materials with both good strength and toughness have been developed [18], [19], [20], [21]. Amongst a variety of animal taxa, eight structural features in biological materials have been identified as the most common: layered, fibrous, helical, gradient, tubular, sutural, cellular, and overlapping [2]. These structural elements can serve as a new paradigm and toolbox to rationalize the complicated mechanical behaviour of biological structures and systematize the biomimetic designs of structural biological materials. Layered structures are often employed to improve the strength and toughness of brittle materials [18]. For instance, cortical bone, a natural material with light weight and high strength, has a lamellar structure [3]. The osteons in cortical bone possess a multi-layered concentric circular structure assembled by collagen fibrils and hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂) nanocrystals [22,23], which contribute enormously to the high mechanical properties and nutrition delivery (Fig. 1a) [24,25]. Similarly, nacre, consisting of 95 vol% brittle aragonite tablets and 5 vol% chitin fibrils and proteins, is the “gold standard” for biomimetic materials. Nacre exhibits high strength (70-100 MPa) and toughness (4-10 MPa∙m^1/2) three orders of magnitude greater (in energy terms) than that of either biopolymers or minerals [18,26]. The mechanical properties of nacre result from its typical hierarchical micro/nanoscale architecture, including the classic “brick and mortar” multi-layered structure and the interfacial interactions between the organic layers and aragonite tablets (Fig. 1b) [27,28]. Such a strategy has been used to develop various synthetic nacre-mimetic materials with improved mechanical properties [16,[29], [30], [31], [32], [33], [34]]. Thus, constructing hierarchically layered architectures in biological materials is a promising strategy to achieve optimal mechanical properties.

Large bone regeneration remains a challenge in clinical applications, especially for load-bearing bones. The most common method to repair bone defects is to implant bone substitutes [35], [36], [37]. For the regeneration of large bone defects, especially for load-bearing bone regeneration, an ideal bone substitute must have good biological properties (biocompatibility, osteogenesis, osteoconduction, and osteoinduction), porous structure, and good strength and toughness. However, most bone substitutes cannot satisfy these requirements, which limits their application for bone regeneration. Although metallic biological materials have enough mechanical strength, their insufficient bioactivity remains a major problem in clinical applications [38]. In terms of non-metallic biological materials, bioceramics have low toughness, and hydrogels have low strength [39,40]. These materials generally have high bioactivity but insufficient mechanical strength. The compressive strength of most conventional bioceramic scaffolds for bone regeneration is lower than 50 MPa [41,42]. Furthermore, bone substitutes need good toughness to prevent fatigue fractures for medical device applications. However, ceramic scaffolds are generally prone to brittle fractures (as they have a fracture toughness typically less than 1 MPa∙m^1/2) [43]. The compressive strength and toughness of cortical bone can reach 100 MPa and 1.2 MJ/m³ [44,45]. In our previous study, we prepared a CaSiO₃–based composite with hierarchical layers inspired by nacre. This biomimetic composite has high mechanical strength, but its osteogenic and angiogenic capabilities are not optimal for bone regeneration [46]. An ideal biological material with outstanding mechanical strength and good osteogenic and angiogenic bioactivities still needs to be systematically explored.

In this study, we considered two factors when designing new materials: chemical composition and nano/microstructure. Graphene oxide (GO) and chitosan (CTS) were used as ideal candidates for the “brick” and adhesive “mortar” in the prepared materials system, respectively. Additionally, HA was introduced to improve the bioactivity of the material. Inspired by the micro/nanoscale structures of nacre and cortical bone, HA/GO/CTS scaffolds with a “brick-and-mortar” layered structure and a multi-layered concentric circular macrostructure were successfully prepared (Fig. 1c, d). Because these scaffolds were synthesized by mimicking co-biological systems (nacre and cortical bone), we termed these scaffolds co-inspired scaffolds (CIS). These CISs present good mechanical properties and bioactivity that may meet the requirements of load-bearing bone substitutes (Fig. 1e).