Microdamage in biological hard tissues and its repair mechanisms

Abstract

Microdamage often occurs in biological hard tissues which mainly include bone tissue and tooth hard tissue, and it primarily comprises diffuse damage and microcracks. The unique microscopic structures of biological hard tissues directly influence the initiation and progression of microcracks. Mechanical forces, loading methods, macroscopic tissue characteristics, aging-related changes, diseases, and medication factors contribute to the complexity of analysis in studying microdamage of biological hard tissues. A large number of literatures have verified the detection and research methods of microcracks. The mechanisms underlying the absorption and repair of biological hard tissues caused by microdamage are still not completely clear. This article reviews the occurrence and development of various types of microdamage in biological hard tissues from microscopic to macroscopic scales, summarizes research approaches of microdamage, elucidates the mechanisms involved in absorption and repair of microdamage, analyzes existing gaps and controversies in current research findings, and proposes potential directions for future research. The study on microdamage of biological hard tissues is crucial for developing biomimetic materials. Such studies facilitate the prediction, control, prevention, and even restoration of microdamage in these materials.

Introduction

Biological hard tissues include bone tissue and dental tissue. Bones contain cortical and cancellous tissue. Dental hard tissues predominantly comprise enamel, dentin, and cementum. Morphologically, microdamage can be classified into three categories: linear microcracks, diffuse microdamages, cross-hatched pattern microcracks [1, 2]. Linear microcracks and diffuse microdamage are predominant among these three categories, with linear microcracks being especially common [3]. Linear microcracks are small fractures on bone's cross section, characterized by sharp outlines and sizes between 50 to 100 µm [4]. To identify microcracks under a microscope, four criteria must be met: they should be of medium size, which means larger than canaliculi but smaller than vascular channels, have sharp edges surrounded by halos, exhibit deep staining in the sections, and have edges that appear deeper than the central area when the focal depth is adjusted [5]. These microcracks are differentiated from cavities based on their volume and aspect ratio. Microscopically, microcracks are typically identified as thin, planar ellipsoidal fractures with thicknesses in the micron range [6]. Tang and Vashishth differentiate between linear microcracks and diffuse microdamage using the ratio of the damage surface (DS) to damage volume (DV), denoted as DS/DV. Higher DS/DV values indicate a crack-like morphology, whereas lower DS/DV values are associated with a diffuse-like damage pattern. The increase in DS/DV suggests a shift in microdamage morphology to crack-like from diffuse-like and higher DS/DV indicate that more and longer microcracks are being formed [7]. Wang figured out that the true appearance of diffuse damage is a mixture of numerous tiny cracks and disabled osteocyte canaliculi [8]. Microcracks with a cross-hatched appearance were usually found in vertically oriented trabeculae and were often surrounded by an area of diffuse staining [4]. In Wang’s experiment, selftapping titanium cortical bone screws were inserted into dogs’ lateral femoral diaphysis, and three types of microdamage, presenting as diffuse, cross-hatched, and discrete linear patterns, were observed within 1 mm from bone–screw interface [8]. Current research predominantly focuses on linear microcracks and diffuse microdamage [9]. The size and shape of microdamage result from a combination of microstructural and macrostructural tissue characteristics, local strain, and other factors such as aging, bone diseases, systemic disease, drug-related factors, or mechanical loads applied to biological hard tissue [10]. Goff reported that under fatigue loads, the initiation and propagation of microdamage are primarily governed by tissue material properties and tissue heterogeneity rather than stress concentrations associated with microscale geometry [11]. At the microscopic level, bone is a complex, heterogeneous, multiphase, and anisotropic layered composite material. Microcracks interact with the microstructure of bone. The occurrence of crack deflection or penetration during crack propagation also hinges on the mechanical properties of interfaces [12]. The microstructure of cortical bone includes bone units, Haversian canals, interstitial bone plates, cement lines, lacunae, and canaliculi. Similarly, cancellous bone exhibits plate-like and rod-like structures, interstitial tissue, blood vessels, lacunocanalicular porosity, resorption cavities, and alterations in the direction of lamellae within trabeculae. Enamel microstructure comprises enamel rods, interprismatic enamel, outer enamel, and inner enamel, while dentin microstructure encompasses intertubular dentin, peritubular dentin, and dentinal tubules. These regions may serve as sites prone to microdamage formation or influence the coalescence, division, and deviation of microdamage. However, some scholars believe that microcracks are influenced not only by anatomical location but also by varying loading conditions [13]. For example, microcracks in dental hard tissues are mainly caused by clinical treatment loads [14].

In 1960, Frost first reported the existence of typical linear microcracks in bone and developed the"bulk staining technique"for observing basic fuchsin-stained microcracks under a microscope which was subsequently widespreadly accepted [15]. Then, researchers refined staining solutions and adopted diverse microscopy techniques to enhance observer precision. However, the invasive, destructive, two-dimensional and labor-intensive nature of these methods prompted the development of non-invasive three-dimensional detection techniques such as micro-CT [14]. In addition, digital technologies have been increasingly utilized for simulating microdamage generation and propagation, though their applicability to in vivo studies remains limited. Recently, the latest research has reported the progress in detection technology for microdamage in synchronous images of human samples [16].

Presently, studies associated with mechanisms of absorption and repair resulting from microdamage focus more on bone tissue than dental hard tissues. The absorption and repair mechanisms of microcracks are different from diffuse microdamage [9]. The mechanisms of absorption and repair of biological hard tissues caused by microdamage are not completely clear, which is the challenge and difficulty of future research which is the challenge and difficulty of future research.The outline of the content discussed in this paper is shown in Fig. 1.

Fig. 1.

Comprehensive diagram of the article

Association between microscopic anatomical structure of biological hard tissues and microdamage

At the microscopic level, biological hard tissue is a complex, heterogeneous, multiphase, and anisotropic layered composite material. The naturally varied microstructures of different biological hard tissues influence the initiation and progression of microdamages in distinct ways. These intricate biological microstructures offer valuable guidance for the research and development of materials and enhancing their fracture resistance. Data on the fracture behavior of dentin can be utilized to design advanced materials. The microscopic structural figures of cortical bone, trabecular bone, enamel, and dentin are shown in Fig. 2.

Fig. 2.

Microscopic structural figures of cortical bone, trabecular bone, enamel, and dentin

Cortical bone

Cortical bone is located mainly in the shafts of the long bones, while a minor proportion is found in the thin external shell surrounding the cancellous bone of the epiphysis and flat and irregular bones [17]. Cortical bone is compact and dense, with a porosity of 5–10% [18]. The osteon, which serves as the fundamental structural unit of cortical bone, comprises mineralized bone matrix organized into concentric cylinders, surrounding a central canal housing neurovascular channels, known as Haversian canals. Under a microscope, the osteons are arranged parallel to the longitudinal axis of long bones, with interstitial bone plates located between adjacent osteons. Cement lines are present at the boundary between osteon and interstitial layers [19]. Cell bodies of osteocytes are situated within ellipsoidal spaces known as lacunae, and osteocyte cell processes extend from these lacunae into slender, elongated channels (0.15–0.55 μm), which are commonly referred to as canaliculi [20].

The pattern of cortical bone destruction correlates with cortical bone’s hierarchical tissue structure, facilitating understanding at the microscopic scale. Microstructures such as osteons, Haversian canals, osteocyte lacunae, interstitial lamella, canaliculi and bone cement line may affect cortical bone’s mechanical behavior [21–23].

In a seminal paper published in 1962, John Currey first reported that microstructural features of bone can serve as stress concentrators and can initiate microdamages [24]. This report also laid the foundation for comprehending how microdamages to bone tissue might influence bone properties. In cortical bone, cracks preferentially develop in interstitial bone, because interstitial bone shows less ductile behavior and has a higher degree of mineralization [25, 26]. When a microcrack in the interstitial bone aligns with the compressive load, it penetrates the osteon parallel to the load [27]. As interstitial cracks enter osteon, they initially encounter the cement line, which is the outermost surface of the osteon and is considered as a decreased mineralized weak interface [28]. Haversian canals may provide stress concentration for the initiation of primary or secondary crack propagation [29]. Similarly, bone cell lacunae serve as strain and stress amplifiers [20]. Ebacher’s study showed that in the bulk of transversely compressed bone, multiple short circumferential cracks initiated at the canaliculi [22]. Their study showed that there are three main forms of microcracks around osteons: interstitial cracks which initiate between interstitial lamellae and may extend into the osteon; radial cracks which initiate from or outside of Haversian canals and penetrate the osteon; annular cracks which located at annular lamellae that are primarily initiate from oval-shaped lacunae [22].

Extensive research has been conducted on the role of microarchitecture of cortical bone in preventing or redirecting the development of microcracks. Both cement lines [30] and osteocyte lacunae [21] have been shown to potentially delay or inhibit the onset and propagation of microcracks. The presence of cement lines can induce a deviation in the trajectory of crack propagation, thereby requiring the expenditure of additional energy and enhancing the material's fracture resistance [31]. A comparative model was developed to further elucidate the toughening mechanism of the cement line, focusing on variations in thickness and elastic modulus of the weak interface, a weak interface is a location where cracks are more likely to occur [32]. The findings reveal that as the interface's strength decreases and its thickness increases, the fracture resistance of the cement line enhances [31]. This protective layer serves as a weak interface, providing valuable insights for the design of related high-performance biomimetic materials. More detailed investigations have revealed that cement lines will arrest crack propagation if their critical energy release rate is lower than that of both the osteon and the adjacent interstitial matrix [33]. Maghami suggests in the study simulates in glycated bone with high advanced glycation end-products (AGEs) using a phase-field framework that cracks trapped in cement lines can prevent further crack growth under cyclic loading [34]. However, if the cement line is tougher than both interstitial matrix and osteons, it will no longer deflect or inhibit the progression of cracks [33]. Phase-field finite-element simulations of microstructural bone indicate that osteocyte lacunae can affect the initiation and propagation of microcracks [21]. The osteocyte lacunae exhibit different responses to load applied in vertical and parallel directions. Under tensile load, osteocytes aligned perpendicular to the load direction are more prone to microdamage, while the opposite is true for compression [35]. If the perilacunar tissue, which develops from the local remodeling processes of lacunar-dwelling osteocytes, is tougher than surrounding tissue, the perilacunar zones may delay or prevent the formation or growth of certain microcracks, despite the stress-concentration effects of the lacunae themselves [21].

The capacity of the microstructure to inhibit crack propagation is affected by bone age-related changes and the length of the cracks. As age progresses, there is a reduction in bone remodeling [36], and there is an increase in both quantity of older osteons and the proportion of potentially highly mineralized cement lines [37]. Consequently, the efficacy of cement lines in inhibiting crack propagation diminishes as age progresses. Aging-induced changes result in enlarged Haversian canals (HC) and a reduction of osteon wall thickness (OWT), which diminishes their ability to deflect cracks and causes fragility fracture [38]. Studies indicate that in the group of the elderly, cracks initially propagate along cement lines and subsequently penetrate through the osteon [38]. In contrast, in the group of young people, cracks merely extend follow cement lines [38]. In addition, it was reported that whether microcracks penetrate or deflect depends on the length of the microcrack. Cracks of average length about 100 μm are usually blocked by cement lines, but longer cracks either deflect (100 to 300 μm in length) or cross the cement line (crack > 300 μm in length), whereby they can be blocked by Haversian canals or continue to grow into adjacent osteons [26].

Cancellous bone

Cancellous bone and cortical bone share similar chemical compositions, yet they exhibit distinct differences both structurally and microscopically [39]. The trabecular architecture of cancellous bone is characteristically loose and has a porosity of 50–90% [40].

The microstructure of cancellous bone is composed of a network of individual trabeculae, which can be classified as either plate-like or rod-like, playing different roles in the formation of microdamage and mechanical properties of cancellous bone [41]. In plate-like trabeculae diffuse microdamage is more dominant, whereas rod-like structures are associated with the formation of linear microcracks [42]. Typically, plate-like trabeculae align along the principal axis of trabecular bone, supporting the majority of mechanical loads, while rod-like trabeculae primarily align transversely, making a smaller contribution to both stiffness and strength [43]. Microcrack in trabecular bone is a significant factor contributing to the mechanical deterioration and remodeling of trabecular bone [44].

In analysis of interaction between linear microcracks and the microstructure of cancellous bone, Mohsin and colleagues identified that linear microcracks tend to follow the trajectory of collagen fibers and were observed to bifurcate when encountering a change in the direction of fibers [45]. In addition, Mohsin and colleagues discovered numerous microcracks that are either perpendicular or at an angle to collagen fibers’ direction within trabeculae, as well as many that create a bridging pattern. Upon encountering microstructure, such as blood vessels, lacunocanalicular porosity, resorption cavities, as well as alterations in the direction of lamellae within trabeculae, microcracks were observed to show deflection or splitting [45]. The process of bone remodeling in trabecular bone can result in the generation of resorption cavities which may diminish the stiffness and strength of trabecular bone. In addition, resorption cavities can serve as stress risers and promote the occurrence and propagation of microdamage [46–48].

In cancellous bone, microdamages predominantly occurs around the center of trabeculae, far away from the trabecular surface in compression tests [11]. This is likely because the interstitial tissue in cancellous bone concentrates in the central areas of the trabeculae and remotes from surface [49]. Furthermore, it has been found that interstitial bone tissue exhibits increased tissue age, decreased toughness [50], is stiffer and harder [51], along with a heightened risk of microdamage. Mohsin [46] and colleagues also reported that microdamage in cancellous bone exists in the older intertrabecular interstitial bone and is oriented longitudinally. This could be due that bone remodeling in cancellous bone predominantly takes place on trabecular surfaces, which positions younger tissue closer to trabecular surfaces and older interstitial tissue at the core of the trabeculae [52]. However, some scholars argue that local tissue stresses increase near the bone surface due to the bending and torsion experienced by individual trabeculae during apparent compression, leading to a higher occurrence of microdamage in areas closer to surface [53].

Enamel

Enamel represents the most highly mineralized tissue found in the human body [54]. Fibrous hydroxyapatite nanocrystals are organized into enamel rods, which constitute the fundamental structural units of enamel. Regions between enamel rods are termed as interprismatic enamel and are characterized by a high protein content [55]. The enamel rod is generally oriented at a right angle to the dentin enamel junction, and its effective diameter progressively broadens as one moves from the dentin enamel junction towards the outer surface [56]. The outer enamel exhibits straight prisms, in contrast to the inner enamel, which displays an oblique crossing pattern of prism bundles [57].

The propagation of fatigue cracks in enamel is predominantly aligned with the orientation of the prisms and is largely restricted to the interprismatic enamel [54]. In the outer enamel, where prisms are more aligned, the propagation of cracks often follows weaker prism boundaries, tend to form microcracks preferentially around these rods when an indentation is made on the axial section of the enamel. Within the inner enamel, the crack path frequently intersects with obliquely positioned prisms, leading to deflections either in-plane as a twist of up to 45° or out-of-plane as a curve of up to 70° [57].

The intricate microstructure of the prisms significantly enhances its inherent ability to impede the progression of cracks [54]. The outer enamel showed a scant improvement in crack resistance with crack extension, indicating a nearly flat R-curve. Mechanistically, neither hydroxyapatite nor the outer enamel possesses the structural attributes capable of dispersing fracture energy as observed in the decussating zone. Enamel's guidance of cracks to the inner region through its straight outer prisms is crucial, which prevents enamel breakage and ensures the cracks localized within the dental structure [57]. Bajaj and colleagues’ studies on human tooth enamel's R-curve behavior demonstrate that crack growth resistance increases with the crack progression from the outer enamel to the inner enamel, as indicated by a rising R-curve. The toughness increment is a function of distance from the Dentin Enamel Junction (DEJ). The apparent toughness of the outer enamel is the lowest (0.67 ± 0.12 MPa·m ^0.5), and the growth toughness of the inner enamel rises from 1.13 MPa·m ^0.5/mm to 3.93 MPa·m ^0.5/mm [57]. Enamel exhibits exceptional fracture resistance due to its microstructure, providing insights for the development of enhanced biomimetic materials. Studying the micromechanism of fracture in dentin can guide the production of adhesives for bonding restorative materials [58].

Dentin

Dentin is a biocomposite material, consisting of intertubular dentin (ITD) enriched with soft proteins and peritubular dentin (PTD) that is stiff and enriched with minerals, featuring tubules that stretch from the dental pulp to the DEJ [59]. The material characteristics of PTD and ITD [60, 61], variations in PTD thickness and diameter of dentinal tubules [62], dentin porosity [58], as well as changes in tubule filling [63], are critical factors influencing the initiation and progression of microcracks within dentin.

First, the influence of the microstructural characteristics of dentin is considered with respect of the occurrence of microdamage. The properties, including increased density, enhanced brittleness [60], higher stiffness, lower cohesive strength, reduced toughness of PTD than ITD, and larger friction angle and weaker strain hardening observed in ITD than PTD, contribute to the increased susceptibility to microcrack formation within PTD [61]. Consequently, microdamage is more prone to occur in PTD. Eltit et al. utilized a bending test on dentin to analyze the process of microcrack formation, indicating that dentinal tubules act as focal points for stress concentration, facilitating the nucleation of microcracks in PTD which subsequently propagate into ITD [64]. An’ investigation on dentin's fracture behavior which employed a plasticity model integrated with a local fracture criterion, revealed that a decrease in tubules’ diameter, PTD thickness and PTD toughness significantly contributes to an increase in microcracks within PTD [62, 65]. In peritubular dentin, microcracks align parallel to the tubules, whereas in the intertubular regions, they intersect perpendicularly with the dentinal tubules [60]. The diameter of tubules proximal to the DEJ is smaller than that near the dental pulp. In addition, tubule density increases as the distance from the DEJ grows. The distinctive composite microstructure results in significant difference in the mechanical behavior between the inner and outer dentin [59]. Maghami and colleagues have demonstrated that the increase in the number of tubules from the outer to the inner dentin leads to an augmentation of microcracks, irrespective of any variations in material properties [62]. Consequently, microdamages begin earlier in the inner dentin than in the outer dentin.

Second, the influence of the microstructural characteristics of dentin on the development of microdamage is considered. Geometric features within the microstructure of dentin, such as PTD, PTD/ITD interface, and the stochastic distribution of dentinal tubules, influence the trajectory of crack propagation. In addition, the amount of energy absorbed during crack propagation exhibits variability [62]. Eltit and colleagues used four-point bending tests to study the propagation of cracks through the dentin. They observed that some microcracks nucleated from the tubules were halted at the PTD/ITD interface [64]. Wang et al. also demonstrated, using a finite element model, that PTD has a shielding effect on the growth of microcracks [58]. PTD which is harder, thicker, and has a higher modulus increases the fracture toughness of dentin and enhances the shielding effect, thereby significantly protecting against the propagation of microcracks. Similar to the behavior observed in cortical bone that the osteon tend to deviate or halt microcrack propagation, models with higher tubule density exhibited crack deviation, microcracking, and crack bridging. Crack deflection was specifically observed in the regions where tubules are close to the crack tip [62]. Outer dentin exhibits higher toughness compared to inner dentin [62], and has a higher tubule density [63]. Microcracks in PTD can result in uncracked ligament bridging, and cracks in inner dentin tend to deflect more frequently [62, 63].

With aging, advanced glycation end-products (AGEs) accumulate in dentin, accompanied by mineral deposition. Non-enzymatic advanced glycation end-products crosslinks accumulate within dentin, specifically within the intertubular collagenous matrix and dentinal tubules of aged dentin, causing the dentin collagen to become more fragile and rigid, thereby increasing the risk of fractures [66, 67]. Mineral deposition leads to a gradual reduction in the diameter of dentinal tubules, eventually resulting in their complete occlusion [68], and decreased porosity [69], which cause an increase in the modulus [59, 70], thickness [70], and hardness of PTD [59, 70], while decreasing its strength [71], and fracture toughness [69], resulting in significant morphological and mechanical changes in dentin [72]. As dentinal tubules become filled, microcracks in PTD decrease within the dentin microstructure, increasing the likelihood of cracks propagating into the matrix, forming relatively straighter cracks and inhibiting toughening mechanisms, such as crack deflection and microcracking [63].

Summary of this chapter

This chapter systematically explores the relationship between the microscopic anatomical structures of various biological hard tissues (such as cortical bone, cancellous bone, enamel, and dentin) and their mechanisms of microdamage (generation, propagation, and repair). The key insight is that the microscopic structure of these tissues, through interface effects (such as crack blocking or deflection), energy dissipation mechanisms (such as crack deflection or bridging), and the inherent heterogeneity of the materials, collectively governs the behavior of microdamage. This structural–function relationship provides important insights for designing biomimetic materials with high damage tolerance. In addition, factors such as age, disease, and medication alter the tissue's microenvironment (such as mineralization, collagen cross-linking, and lumen filling), further influencing the accumulation and repair efficiency of microdamage, highlighting the importance of multi-scale research in understanding the mechanical properties of bones and teeth.

Generation of microdamage in biological hard tissues

The morphology of microcracks, influenced by the microanatomical structure of the tissue, mechanical stimulation, age-related changes, diseases, and pharmaceutical factors, is the combined result of local strain and tissue characteristics. Microdamage occurs and accumulates under physiological and pathological conditions [15].

Mechanical stimulation

Loading conditions affect the formation and behavior of microcracks [13]. The load encompasses the total forces exerted on a substance. Common types of stress include compression, tension, shear, bending, torsion, and off-axis shear. Compression, tension, and shear are uniaxial stress mechanisms, whereas bending, torsion, and off-axis shear are more complex, involving forces applied on two or more planes [73]. Furthermore, multiple types of stress may be applied simultaneously, adding to the complexity of biomechanical analysis. The force exerted can influence mechanical strain, which in turn modifies cellular activity. The force exerted can influence mechanical strain, which in turn modifies cellular activity. The principal mechanisms of bone and cartilage damage are shown in Fig. 3 [73].

Fig. 3.

Principal mechanisms of injury of bone and cartilage are illustrated [73]

Different mechanical stimuli lead to various types of microcracks. Under mechanical stimulation, areas subjected to tensile strain in hard tissues exhibit low-intensity diffuse microdamage. In addition, diffuse damage primarily occurs in trabecular bone tissue subjected to pure tensile stress. In contrast, areas subjected to compressive strain and internal shear forces exhibit high-intensity linear microcracks [9, 31, 74]. Static (creep) loads tend to produce diffuse microdamage, whereas cyclic loads are prone to inducing linear microcracks [75]. In addition, with the increase of cyclic loading, the microdamage regions grow within osteons rather than forming new damage sites [74]. In addition, under cyclic loading, diffuse damage manifests earlier than linear microcracks [75, 76]. Because compared to linear microcracks, diffuse microdamage is more likely to occur and does so at lower load levels. In cyclic loading, the intensity of loading, along with its frequency (f) and the number of cycles (N), are essential factors that impact bone fatigue damage [77, 78]. With the increase in loads and the number of strain cycles, the operational microdamage threshold of bone is surpassed, resulting in the accumulation of microdamage. This accumulation leads to shear failure at the cement lines of the osteon and collagen, ultimately causing the formation of microcracks [79, 80]. Linear microcracks were most prevalent at intermediate load cycles (2100 cycles) but diminished at higher load cycles (3000 cycles), potentially due to being obscured by diffuse damage [74]. Frost reported that when the deformation of the bone surpasses 1500 microstrains (0.15%), osteoblasts are triggered to generate additional bone mass [81]. Conversely, at a strain level of 300 microstrains (0.03%), the basic multicellular unit (BMU) that includes both osteoclasts and osteoblasts is activated to resorb bone tissue.

In bone, microcracks mainly result from the physiological loads and mechanical stress experienced in daily life. Under normal physiological conditions, human bones primarily endure compressive, bending, tensile, and torsional loads, whereas the jawbone is predominantly subjected to cyclic loading, distinguishing it from other bone tissues in the body. Li et al. developed a model of exercise-induced fatigue injury in the rat ulna [82]. They found that in the early stages of strenuous exercise, such as 3 days for rats or 1 month for humans, rapid microdamage, reduced fatigue mechanical properties, and significant bone resorption were examined [82]. Jawbone may sustain microdamage under fatigue loading [83]. Research on microcracks in relation to the jawbone reveals that the jawbone is essentially subjected to rhythmic cyclic loads during chewing, experiencing high-amplitude bursts during meals and low-amplitude bursts at other times [84, 85]. Furthermore, orthodontic loads during treatment create additional mechanical disturbances, causing further accumulation of microdamage in the alveolar bone [83]. Quantitative analysis of microcrack density during orthodontic treatment indicates significant variability in microcrack distribution across different sides, locations, and treatments [86]. However, the study by Präger et al. suggested that microdamage in the alveolar bone caused by orthodontic treatment manifests as microcracks and diffuse damage, and is independent of the manner and duration of load application [83].

Microcracks in dental hard tissues are predominantly induced by mechanical stimulation during dental procedures [87]. Microcracks in hard dental tissues may occur in the enamel, dentin, and cementum. In vitro study results indicate that there are no pre-existing dentinal microcracks in the dental hard tissues of teeth that have not undergone endodontic treatment [88]. During root canal treatment, various factors such as the taper of root canal preparation instruments, movement methods of instruments, root canal preparation techniques, design of root canal cutting files, duration of preparation time, and material of root canal preparation instruments can all lead to varying degrees of dentinal microcracks [89–93]. Interestingly, a recent micro-CT in vitro study published in 2022 suggested that chemomechanical instrumentation of the root canal system generally does not cause dentinal microcracks. Instead, the anatomy of the dental root and root canal are the primary contributing factors [94]. This study was the first to refute the previous findings that chemomechanical preparation causes microcracks. The cementum on the surface of the tooth root is similar to bone tissue. A common complication in orthodontic treatment is root resorption, which may be associated with the stress generated during orthodontic tooth movement. This stress can induce and propagate microcracks in the root and surrounding bone tissue. In addition, both the root and the periodontal ligament exhibit creep behavior [95, 96], which can accelerate the formation of microdamages on the root surface during orthodontic treatment. Xiao et al. analyzed the relationship between orthodontic force and the development of microcracks on the tooth root surface. They observed that microcracks usually formed on the compressed side of the tooth apex, and they found that the number, width, and length of these microcracks were positively correlated with the magnitude and duration of the applied orthodontic force [97]. The results of finite element analysis indicate that improper orthodontic forces can directly induce the formation and propagation of microcracks and may contribute to further root resorption. The region of peak stress at the apical region is consistent with the microcrack region observed via Scanning Electron Microscopy (SEM). Various load magnitudes initiate microcracks at varying rates, and the propagation of microcracks differs under varying loading directions [97]. Therefore, proper intervals and orientations of orthodontic forces can help minimize microcracks and prevent root resorption [97]. If the correlation between orthodontic forces and microcracks, as well as the potential association between microcracks and root resorption, can be established, current orthodontic strategies and techniques could be refined to prevent root resorption.

Tissue characteristics

Protein levels influence the formation of microcracks by affecting tissue properties, and a reduction in protein levels may lead to an increase in microcracks. Studies have found that mice fed a diet high in saturated fat (HSF) exhibit increased formation and propagation of cortical microcracks [98]. A polyunsaturated fat diet high in ω−6 (PUFA) promotes protein synthesis and collagen function, improves the intrinsic properties of bone, and limits the formation of microcracks [98]. Mice on the HFD50:50 diet (50:50 mix of saturated and unsaturated fats, high in ω−9) showed smaller microcracks compared to the control group [98].

In addition, it have also shown that high-fat diet (HFD) and atherogenic diet (AD) cause bone microstructure damage (like osteoporosis) and vascular calcification by inducing unbalanced differentiation of bone marrow stromal cells (BMSCs), inflammatory responses and abnormal lipid metabolism. An isoflavone glycoside called Caviumin β-D-glucopyranoside (CAFG) effectively reverses these lesions by regulating the osteogenic/osteoclastic balance, inhibiting inflammation, and improving lipid metabolism [99].

Changes in mechanical strength and other parameters suggest that the proportion of dietary fatty acids may affect the ability of bone to resist microdamage by changing bone tissue microstructure (e. g., bone density, porosity) and mechanical properties (e. g., bending and torsion resistance) [100].

The structure of cortical bone is more conducive to the growth of microcracks than that of cancellous bone [1]. Cancellous bone exhibited a significantly higher microcrack density compared to cortical bone, while cortical bone had a significantly longer microcrack length [1].

Age-related changes

Studies indicate that microcracks are not associated with sex [101], and their occurrence increases exponentially with age [102]. Contrarily, earlier research reported no significant correlation between age and microdamage [103]. These differing results may stem from variations in sampling locations and mechanical loading conditions. The changes of linear microcrack density and diffuse damage density with age of human vertebral cancellous bone donor are shown in Figs. 4 and 5, respectively [102].

Fig. 4.

Variation of linear microcrack density as a function of donor age in human vertebral cancellous bone [102]

Fig. 5.

Variation of diffuse damage density as a function of donor age in human vertebral cancellous bone [102]

Age influences microcrack morphology, making it predominantly linear in the elderly [104]. Microdamage accumulation, rising with age as linear microcracks, is a crucial factor in bone quality, contributing to fragility and increasing fracture risk in the elderly [105]. The capacity of bone tissue to form diffuse damage diminishes with advancing age [106]. Younger individuals tend to form more diffuse damage, whereas older individuals tend to experience more microcracks [106]. A study indicated that bones from elderly donors (83 ± 3 years) contained more linear microcracks in the cortex, whereas bones from younger donors (40 ± 10 years) exhibited a greater extent of diffuse microdamage [104]. After root canal preparation, microcrack propagation in young teeth tends to occur through the dentinal tubules, whereas in older teeth, it tends to deflect towards the periphery of PTD [107]. Furthermore, physiological loading levels periodically induce microdamage, and serum exosomes from younger individuals enhance the osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMSCs) to repair bone microdamage [108]. With advancing age, the number of BMSCs remains constant, while the number of mature osteoblasts declines, indicating that the osteogenic differentiation ability of BMSCs deteriorates over time, leading to impaired repair of microdamage. The ability of BMSCs to repair bone microdamage diminished with age, leading to an accumulation of microdamage and a higher risk of fractures [109]. In addition, the presence of long microcracks in elderly individuals indicates a decreased capacity to repair these microcracks [1].

Disease and medication factors

According to recent studies, diseases such as osteoporosis, diabetes, and osteoarthritis, osteogenesis imperfecta, cancer, as well as the use of bisphosphonates, are associated with the occurrence and progression of microcracks [110]. The targeted bone remodeling process usually repairs microcracks mediated by osteocyte apoptosis [109]. However, these diseases and the use of these medications may disrupt the balance between the formation and repair of microdamage.

Osteoporosis is a significant public health concern among the elderly, characterized by reduced bone mass, increased porosity and bone microdamage caused by fatigue load [108]. Moreover, studies conducted in vivo revealed that in osteoporosis mice, the process of targeted bone remodeling mediated by osteocyte apoptosis becomes insufficient to repair microcracks, resulting in the accumulation of microcracks [111]. Findings from animal experiments show that serum exosomes from young rats with overexpression of miRNA-19b-3p can enhance the osteogenic differentiation capacity of BMSCs in aged osteoporotic rats following mechanical stress, providing a novel therapeutic strategy for the repair of bone microdamage and the prevention of fractures [108]. Ciarelli examined biopsies collected from vertebral fracture patients and healthy individuals with high or low bone formation rate and pointed out that lamellar structure has been hypothesized to guide microcrack propagation in order to optimize bone strength and toughness, and osteoporotics with fracture had less pronounced lamellation than healthy normal and may be more prone to fracture [112].

Type 1 and type 2 diabetes (DM1 and DM2) lead to alterations in bone microarchitecture, affecting stress distribution within trabecular bone [113]. This in turn influences the locations and quantities of microdamage accumulation within the bone microstructure [113]. Impaired stress relaxation in the glycated bone matrix can lead to high-stress-concentration areas, resulting in microcrack coalescence and increased microcrack length [114]. These changes result in altered cracking behavior and reduced propagation toughness in glycated bone, promoting the generation and accumulation of microcracks [114]. In the later stages of diabetes, microcracks accumulate and extend, compromising the mechanical properties of bone and eventually leading to fractures [45]. A 2020 histomorphological analysis by Liu et al. [115] first demonstrated that high glucose conditions lead to the accumulation of numerous submicron microcracks in bone tissue, significantly increasing the spatial accumulation density of linear microcracks and diffuse microdamage. Linear microcracks (LM) predominate in glycated bone. Severe osteocyte canaliculi damage occurs around linear microcracks [116]. Immunostaining shows diabetic patients have significantly higher densities of both linear microcracks and diffuse microdamage compared to non-diabetic individuals, with notable accumulation [116]. Under high glucose, every critical stage of bone-targeted remodeling is impaired: increased apoptosis of"bystander"osteocytes, damage to microtubule integrity, reduced RANKL secretion, decreased osteoclast recruitment and bone resorption, and impaired osteoblast-mediated bone formation [116]. These disruptions promote accumulation of more severe linear microcracks, leading to bone degradation and increased fragility [116].

Microcracks frequently occur in areas with significant cartilage calcification, particularly in the subchondral bone and calcified cartilage [117], which may be associated with osteoarthritis [118]. Linear microcracks have been found in osteoarthritis (OA) patients with an average age of 67 [1]. Microcracks in subchondral bone may contribute to the degeneration of joint cartilage by facilitating vascular invasion into the calcified cartilage, which in turn promotes cartilage ossification and leads to the thinning of the joint cartilage [119]. Evidence suggests that the occurrence of subchondral microcracks can enhance osteocyte density, which in turn regulates subchondral remodeling and mitigates cartilage damage [120].

Osteogenesis imperfecta (OI) may lead to an increased microdamage in bone. OI is a genetic disease of collagen or collagen-related proteins that adversely impacts bone mass and fracture resistance [1]. Davis reported that Brtl/+ mouse model for OI is more susceptible to microdamage accumulation than age-matched WT [121]. Nakayama found out that the c-src knockout mice, which also develop OI, had increased numbers of microcracks [122]. Dong figured out that these changes may reduce the bone's ability to form diffuse damage regardless of whether it is under tensile lateral force or compressive lateral force, ultimately contributing to the tissue's increased brittleness [123]. Iwata reported that accumulation of microdamage and low bone mass in the femoral head is a cause of subchondral insufficiency fracture in a patient with OI [124].

Cancer, metastatic cancer or post-cancer radiotherapy may cause microcracks. Rabelo did a histological and microcrack analysis using bone samples from twenty patients who underwent some mandibular bone removal as part of the treatment of oral squamous cell carcinoma and reported that microcracks were present in the proximity of the tumor in seven patients and in the surgical margin in four patients [1]. Therefore, Rabelo pointed out in this experiment that the mandible cortical bone microarchitecture changes in the proximity of the squamous cell carcinoma lesion [125]. Metastatic cancer, which constitutes the majority of tumors found in bone, often spreads to the spine, significantly compromising bone quality [1]. In cases of osteolytic and mixed metastatic disease, load-induced microdamage is markedly elevated, with diffuse cross-hatched microdamage, linear microdamage, and microfractures observed in metastatic tissue, further highlighting the deterioration of bone integrity [126]. Cancer survivors treated with radiotherapy experience direct bone damage and increased fatigue-induced microdamage, as irradiation reduces bone fatigue strength and increases creep damage [127].

Bisphosphonate (BP) therapy was linked to significantly reduced bone strength, likely caused by the accumulation of microcracks and the absence of noticeable improvements in bone volume or microarchitecture [128]. Bisphosphonates are used to treat osteoporosis and osteogenesis imperfecta in adults and children [129]. BP therapy inhibits bone remodeling and reduces the clearance of microdamage; hence in the early stages of microcrack propagation, the density of bone microcracks rises [130, 131], while the length of microcracks remains unchanged [130, 132]. Prolonged bisphosphonate therapy may decrease compositional heterogeneity, weaken structural integrity [133], and create pathways for microbial colonization [134]. Research showed that bone models treated with bisphosphonates characterized by low material heterogeneity and high microcrack density exhibited higher initial fracture resistance than untreated models which had high material heterogeneity and low microcrack density [130, 131]. Therefore, bisphosphonate therapy can mitigate trabecular perforations and enhance the fracture resistance of bone in the early stages of treatment. However, with the prolongation of treatment duration, this trend is reversed due to the suppression of bone remodeling. Bone remodeling is inhibited, cracks will accumulate as they are not promptly repaired by osteoclasts, thus becoming larger, which significantly increases clinical fracture risk. This lack of repair and subsequent crack propagation can be a key factor in catastrophic fractures [135]. Therefore, for patients on long-term bisphosphonate therapy, predicting the critical timepoint for microcrack accumulation could help to optimize the treatment duration, allowing bisphosphonates to be used long enough to increase bone volume and reduce trabecular perforations while stopping the medication before severe microcrack accumulation occurs [133]. By the way, eldecalcitol (ELD), an active form of vitamin D analog approved for the treatment of osteoporosis in Japan was reported to increase bone mineralization and suppresses bone microdamage [136].

Summary of this chapter

This chapter systematically analyzes the formation mechanism of microdamage in biological hard tissues, revealing that the core issue lies in the dynamic interaction of various factors (mechanical stimulation, tissue characteristics, age changes, diseases, and medications), which disrupts the balance between microdamage formation and repair. This disruption leads to the accumulation of microdamage, ultimately damaging the mechanical properties of tissues and increasing the risk of fractures. This research provides a crucial theoretical foundation for clinical fracture prevention, optimizing treatment strategies (such as load control and timing of drug intervention), and the design of biomimetic materials. Future research should delve into the direct causal relationship between age and microdamage, as well as the development of real-time monitoring technologies in living organisms.

Impact of microdamage on the mechanical behavior

Microdamage influences the mechanical properties of materials. Current perspectives suggest that linear microcracks and diffuse microdamage may lead to the degradation of bone mechanical properties [137, 138], such as reductions in hardness, strength, fracture toughness, and elastic modulus [80, 139–141], along with an increase in brittleness [137]. The formation of microdamage is a critical determinant of bone quality. Accumulation of microdamage in bones weakens bone strength and increases the likelihood of fractures. Therefore, evaluating bone microdamage at different hierarchical levels is vital for a deeper understanding of bone quality and fracture risk [142].

Chapurlat et al. suggest that linear microcracks and diffuse microdamage exhibit distinct biomechanical responses [110]. The loss of elastic modulus exhibits a linear relationship with diffuse microdamage and a quadratic relationship with linear microcracks [50]. Fracture toughness increases as the length of microcracks increases [50]. Regardless of the material's local properties, heterogeneity, and the distribution of microcracks, an increase in microcrack density can enhance fracture resistance, reduce crack volume, and decrease damage energy density [132]. If microcracks are not severe, bones can tolerate a substantial number of them, thereby exhibiting characteristics of a damage-tolerant material [143]. However, some studies indicate that the reduction in toughness is not associated with the accumulation of microdamage [144]. The presence of diffuse damage is regarded as a toughening mechanism, as it dissipates energy and delays crack propagation, thereby preventing bone failure [145]. Diffuse damage enables bones to dissipate energy and delay the onset of fractures, thereby enhancing fracture resistance [104].

Repeated mechanical strain on bones can lead to the formation of microcracks, which, if left unrepaired, may propagate, merge, and ultimately cause catastrophic fractures [146]. Extended Finite Element Method (XFEM) results indicate that microcracks near the major crack tip increase the critical stress intensity factor [24]. The main crack may form from the merging of microcracks [147], and the development of microcracks near a propagating main crack can slow down the crack propagation rate [148]. A higher volume fraction of microcracks observed within a given radius leads to greater energy dissipation and slower main crack propagation [149]. New cracks adjacent to a propagating crack will slow down microcrack propagation, whereas cracks in front of and behind it will accelerate the microcrack propagation [150]. Ultimately, microcracks will coalesce into fatal macrocracks under high strain only when they become very dense [151].

Detection and analysis techniques for microdamage

The traditional and widely applied technique for studying microcracks involves staining tissue sections of hard tissue specimens and observing them under a microscope [15, 152]. This method allows for the quantification of several parameters, including the number of cracks (Cr.N), microcrack density (Cr.Dn), microcrack length (Cr.Le), microcrack surface density (Cr.S.Dn), which enables the assessment of microdamage accumulation [105, 108, 139]. Frost first developed the"bulk staining technique" [15], which involves staining with basic fuchsin and then observing the samples under an optical or fluorescence microscope [153]. In addition, subsequently, Burr modified the staining method to enhance the penetration of basic fuchsin into mineralized tissues [153]. This method stains the sample prior to further processing, allowing for clear distinction between microcracks and processing artifacts generated during specimen preparation. Using fluorescence can help identify more microcracks and enhance the precision of observations [154]. The advantage of fluorescent dyes is their ability to continuously stain cracks in the same specimen following various disturbances, enabling the assessment of crack initiation and growth [152]. Other available microscopy techniques include transmitted light microscopy [155], epifluorescence microscopy [155], laser scanning confocal microscopy (LSCM) [156], backscattered electron microscopy (BSE) [157], stereomicroscopy [158], atomic force microscopy (AFM) [159], scanning electron microscopy (SEM) [160], and transmission X-ray microscopy (TXM) [161]. The same sample was observed under microscopes of different accuracy [152]. The cracks marked with iodine contrast agent are shown in Fig. 6.

Fig. 6.

Micro-CT of bone specimen of cross Section  2 mm  × 2 mm [152]

Traditional methods for imaging and quantifying microdamage accumulation in hard tissues are limited to histological sections, which are inherently invasive, destructive, two-dimensional, cumbersome, and labor-intensive. Moreover, two-dimensional tissue sections are unable to study the accumulation of microcrack spatial changes in volume related to mechanical loading, bone density, and microstructure. Ideally, microcracks in hard tissues should be observed, analyzed, and measured in three dimensions with isotropic and high spatial resolution. Cone-beam computed tomography (CBCT) appears to lack the resolution required to observe microcracks [158]. Micro-CT [152], synchrotron radiation micro-CT (SR micro-CT) [162], and contrast-enhanced micro-CT can non-destructively detect the presence, three-dimensional spatial location, and gradual accumulation of microcracks in vitro [163]. However, the resolution of these devices is insufficient at present. Further development is, therefore, required Additional methods include Nonlinear Resonance Ultrasound Spectroscopy (NRUS) [164], infrared thermography [165], diode-based laser ultrasound [166], sodium fluoride (Na18F) tracer microcrack positron emission tomography (PET) [167], and high-energy monochromatic synchrotron radiation X-ray tomography [168]. The fluorescence photoinduced electron transfer (PET) sensor, proposed in 2003, can quantify microdamage, measure crack propagation, and provide data for modeling the behavior of bone materials. However, no further related reports have been found [152].

Several digital discrete methods are used to simulate crack propagation, such as digital image correlation (DIC) [169], and digital volume correlation (DVC) analysis technique [170]. Finite element analysis is a widely used method for examining microstructural and small-scale effects [171, 172]. Numerous numerical strategies exist for simulating fracture propagation, such as the extended Finite Element Method (XFEM) developed by Moes’ team [173], advanced isogeometric analysis presented by Ghorashi's group [174], and the cohesive zone model crafted by Remmers [175]. Goswami et al. [176]. Have developed a new physics-informed neural network (PINN) scheme to predict crack growth using the phase-field approach. Other methods for simulating crack propagation include the damage evolution method, which employs constitutive algorithms [177] and techniques involving mesh refinement [178]. In 2023, Buccino et al. were the first to apply a convolutional neural network (CNN) to detect bone microstructure and microdamage progression in synchronized images of human samples [179].

Mechanisms of microdamage repair in biological hard tissues

Bone resorption induced by linear microdamage in bone tissue

Unlike dental hard tissues, bone has the ability to self-repair and adapt to the mechanical environment [6, 21]. Frost first proposed that the goal of bone remodeling is to eliminate microdamage, thereby maintaining skeletal integrity [15]. The primary purposes of bone remodeling are: repairing microcracks within the bone, adapting the macroscopic stiffness and strength to meet mechanical demands, and regulating calcium homeostasis [180].

Microdamage is a key factor contributing to bone resorption. Schaffler et al. suggest that microcracks and bone resorption mutually reinforce each other. The accumulation of microdamage results in an increase in resorption cavities, decreased bone strength, increased brittleness, and elevated bone stress and strain, which further accelerates the formation of microdamage [181]. Bone remodeling is widely understood to be triggered by osteocyte apoptosis. The Rank/Rankl/OPG pathway is a critical biochemical process in bone remodeling, regulating the interactions between osteoblasts and osteoclasts [182]. Osteocytes play a crucial role in bone remodeling due to their ability to detect microdamage, which initiates the remodeling process [183]. Osteocytes, as mechanosensitive cells responding to microcracks under mechanical loading, respond to biochemical and biomechanical stimuli and are responsible for transmitting biochemical signals to osteoclasts [180]. By recruiting osteoclasts and osteoblasts, the repair process is initiated to maintain bone homeostasis [184].

Microcracks have been confirmed to cause osteocyte apoptosis, and apoptotic bodies can induce osteoclast formation, thereby promoting the resorption of bone tissue [185]. Osteocyte apoptosis is a critical step in the targeted removal of microcracks and the associated signaling mechanisms through osteoclast-mediated resorption in damaged bone and osteocytes. Substances released from apoptotic osteocytes travel through canaliculi, serving as potential sources of transmissible signals [183]. At microcrack sites, osteocytes undergoing apoptosis can communicate with surrounding living cells by releasing messenger molecules, such as ATP, which act as'find-me'signals [186]. Living osteocytes adjacent to apoptotic osteocytes then release osteoclast recruitment molecules, such as RANKL, to initiate targeted bone remodeling [116]. However, there are differing perspectives on the mechanisms through which microcracks induce osteocyte apoptosis and bone resorption. Jan et al. employed specific cell staining techniques to confirm that relative microcrack displacement can tear adjacent osteocytes, suggesting that this tearing process may lead to the secretion of bioactive molecules into the extracellular matrix, thereby initiating a bone resorption response [187]. However, at the crack tip, due to the smaller relative displacement, no osteocyte rupture was observed. Tami et al. argue that linear microcracks disrupt the transport of local canalicular fluid between osteocytes, resulting in a loss of this fluid transport [188]. This leads to hypoxic stress, causing osteocyte apoptosis [189]. Herman et al. suggest that microcracks directly cut through osteocytes, resulting in their necrosis and subsequently triggering apoptosis in neighboring osteocytes, thereby inducing osteoclast formation [9]. Cardoso et al. discovered that linear microcracks induce osteocyte apoptosis by disrupting the osteocyte network, and they directly control the activation and targeting of the local bone remodeling repair response [190]. Vasquez-Sanchot et al. proposed that cracks formed in bone under compression can generate piezoelectricity, which induces osteocyte apoptosis and subsequently promotes osteoclast activation [191]. Huang H et al. found that microdamage affects osteocyte expression of M-CSF, RANKL, and OPG, thereby regulating osteoclast formation [185, 192].

Li et al. observed that during fatigue loading, with the generation and development of microcracks, the number of empty osteocyte lacunae significantly increases around nearby osteons, and osteocyte apoptosis consistently occurs, leading to targeted bone remodelin [82]. Apart from osteocyte apoptosis inducing the activation and formation of osteoclasts, calcium ions themselves also promote osteoclast differentiation [193, 194]. Vasquez-Sancho et al. discovered the presence of flexoelectric fields around microcracks in bone tissue [191]. The field strength near the apex of these microcracks can reach several kV/m. This intensity can induce osteocyte apoptosis. An electric field of 1 kV/m is sufficient to damage cells, while fields exceeding 10 kV/m can cause immediate cell necrosis [195]. However, recent studies have reported that only the presence of cracks does not induce cell apoptosis; mechanical stimulation of the cracks is necessary to cause cell death [196]. This finding differs from previous studies, necessitating further research for verification. Jiang et al. summarized the key pathological factors of microcracks and fatigue in osteocyte apoptosis and their potential mechanisms, such as causing dendritic process rupture, damaging Cx43 gap junctions, rupturing the plasma membrane, upregulating cellular communication networks, and upregulating CCN2 through the ERK1/2 pathway [197]. Panx1 and P2X7R are required for the initiation of bone remodeling induced by bone microdamage. This mechanism aligns with the signaling pathways involved in localized injury remodeling and apoptosis initiation in other tissues [186]. Pannexin-1 (Panx1) channels do not influence the apoptosis of osteocytes that occurs in response to bone fatigue and microdamage. However, these channels are essential for apoptotic osteocytes to induce RANKL expression in neighboring bystander osteocytes, thereby initiating osteoclast-mediated bone remodeling [186].

Bone remodeling induced by linear microdamage in bone tissue

The stages of bone remodeling include quiescence, activation, resorption, reversal, bone formation, and termination [198]. Typically, normal bone remodeling progresses slowly, with the entire cycle spanning several weeks to months. In 2007, Martin proposed that osteocytes recruit the basic multicellular unit (BMU) to repair microdamage [199]. This is followed by the osteoclasts activation of the BMU, which initiates targeted bone remodeling. BMU serves as the primary biological mechanism for repairing microcracks, facilitating the removal of microdamage and the formation of new bone [6]. Osteoclasts, osteoblasts, macrophages, and various immune and hematopoietic cell aggregates combine to form a transient, mobile structure known as BMU, which focuses on repairing a specific discrete area [198]. In the basic multicellular unit, osteoclasts lead the way for osteoblasts, forming a cylindrical tunnel known as a cutting cone in cortical bone, or creating a trench-like excavation along the trabecular surface in cancellous bone [200]. As they progress through cortical bone, they remove old bone tissue and leave behind new, slightly softer and harder bone, forming osteons. The bone cells deposited by BMU reside in the new bone matrix and occupy spaces called lacunae. These cells contribute to the mechanical sensing behavior of bone through the lacuna–canalicular network [201].

When a new BMU is activated by microdamage, or when an existing BMU is sufficiently close to the microdamage, the advancing BMU enters a network of apoptotic bone cells around the microdamage area [199]. Osteoclasts within the BMU are attracted to these apoptotic cells, releasing strong acids and enzymes to resorb the damaged bone. Subsequently, osteoblasts generate new bone. Together, these cells form a BMU with a diameter of approximately 200 μm, advancing at a rate of about 40 μm per day, and in cortical bone create a new bone unit with a circular cross section termed an osteon [6]. Osteoclasts identify, monitor, and repair microdamage. Bone resorption sites indicate newly activated BMUs on the surfaces of the endosteum, periosteum, or Haversian canals [199]. Microdamage not only target the activation of new BMUs but may also attract or guide existing BMUs to continue moving through the bone matrix. A preliminary analysis of the relationship between the average microcrack length and the density of BMU resorption spaces in cortical bone indicates that the functional area of a BMU is approximately 40 times its actual cross-sectional area [199]. The expected length of crack removed by a single BMU is given by the repair length ΔL = (A_BMU/A_t) *L0, where A_t is the total cross-sectional area of the bone, L0 denotes the initial length of the microcrack present within the bone, and A_BMU indicates the cross-sectional area of a single BMU that passes through this segment at a random location. If the number of BMUs is denoted as Nr, then ΔL = Nr (A_BMU/A_t) * L0. The activation of BMUs to repair microcracks can significantly reduce the likelihood of fractures resulting from the propagation of these microcracks [199].

The activity of BMU and the clearance of bone damage are determined by two factors: the ratio of Bax to Bcl-2 and the differences in their local tissue levels. Bax is a primary effector molecule that promotes apoptosis and is commonly expressed in damaged bone cells. In contrast, Bcl-2 prevents apoptosis and is produced by osteocytes located further from the injury site [202]. The equilibrium between Bax and Bcl-2 confines the apoptotic region to within a few hundred micrometers of the microcracks [203]. Olivier et al. discovered in a rat fatigue test model that the number of osteocytes expressing the pro-apoptotic molecule Bax was highest near microcracks, decreasing with increasing distance, and nearly reaching zero in a nearly linear manner at approximately 2–3 mm away. The number of osteocytes expressing the anti-apoptotic molecule Bcl-2 reached its peak at a distance of 1–2 mm and decreased to the levels observed in the control group at 3–4 mm [203].

The work of Jung, Shu, and Sun et al. demonstrated that microcracks lead to an increased release of calcium ions, with the efflux induced by mechanical force occurring in areas of bone matrix damage caused by this force [204, 205]. Jung et al. quantitatively studied the relationship between microdamage in MC3T3-E1 cells and localized activation of intracellular calcium signals, finding a spatial correlation between the microdamage and the induction of calcium signal activation within these cells [206]. Shu et al. investigated the impact of microcracks on osteoblasts by comparing the cellular responses on hydroxyapatite (HA) specimens, both with and without microcracks [204]. They observed that the presence of microcracks led to increased calcium release, which they proposed might enhance the osteoblast response. Sun's experiments with normal and demineralized bone matrices revealed that the calcium signaling response under mechanical load was reduced in the demineralized bone matrix group [207]. The bone matrix functions as a"mechanochemical sensor"translating mechanical loads into chemical signals that promote osteoblast differentiation and microdamage repair. Furthermore, calcium ions are more concentrated in the confined space between the cell membrane at the adhesion site and the bone matrix than in an open space [207].

Repair mechanisms for diffuse microdamage in bone tissue

Herman et al. noted that, unlike the repair of linear microcracks, the repair process of diffuse microinjuries does not involve the recruitment of osteoclasts triggered by osteocyte apoptosis to initiate directed bone remodeling. There is no observed osteocyte apoptosis or osteoclast activation, and the survival rate of osteocytes remains unaffected. Instead, this repair process leads to random bone remodeling [9]. Instead, it initiates random bone reconstruction [9]. This may be due to the sub-lamellar microcracks in diffuse damage being too small, often less than 1 μm, to compromise the integrity of osteocytes or impair the local transport of fluids and solutes between them. As a result, these microcracks do not induce the metabolic stress that leads to cell death, and thus fail to activate local bone remodeling activities [9]. Therefore, the effectiveness of bone remodeling in eliminating diffuse damage is significantly lower compared to that for linear microcracks [208]. Bentolila et al. reported that diffuse microdamage might be incidentally eliminated by other nearby targeted remodeling sites, but this process is not particularly effective [208]. Bonewald et al. discovered that osteocytes might directly contribute to the repair of diffuse microdamage by regulating phosphate mineralization through PHEX, DMP-1, and MEPE [209]. Yan and Bjela et al. proposed direct mechanical stretching and fluid shear as stimuli initiating diffuse microdamage repair mechanisms [210, 211]. Jung H et al. suggested a theory involving extracellular calcium release (DMICE) through voltage-gated calcium channels (VGCC), increasing osteoblast intracellular calcium (Ca2 +) signaling to initiate anabolic function [212]. Seref-Ferlengez et al. indicated repair might occur through alternative mechanisms not requiring damaged matrix resorption [75]. Boyde et al. highlighted bone's ability for self-repair, distinct from remodeling-mediated repair, involving direct mineral filling of microcracks, potentially repairing submicron cracks and reducing larger ones [75, 213]. Although osteocyte detection and repair mechanisms remain incompletely understood, osteocytes are believed crucial in repair by regulating matrix mineral production [75].

Repair mechanisms for microdamage in dental hard tissue

There has been considerable research on the mechanisms of hard dental tissue resorption, primarily focusing on cementum. In contrast, studies on the resorption of dentin and enamel are limited, and research on the repair mechanisms of hard dental tissues is even scarcer. Current studies propose two potential mechanisms for the resorption of hard dental tissues: one is a bone tissue-like resorption mediated by odontoclasts, and the other is resorption induced by the excessive release of calcium ions.

The tooth root is intimately connected to the alveolar bone, sharing similar physiological and mechanical environments. Consequently, the resorption processes of both the tooth root and the alveolar bone are analogous [214–217]. Cementocytes are analogous to osteocytes [218], while cementoclasts resemble osteoclasts [214], with both cell types exhibiting comparable biological functions. Research has identified two molecular pathways responsible for tooth root resorption. The first is the RANK/RANKL/OPG pathway [219], which primarily facilitates osteoclast formation [220]. The second pathway is ATP-P2X7R-Il1-β [221], which is pivotal in the metabolism of necrotic tissue and the resolution of inflammation [222].

In a rat orthodontic model, calcitonin was found to inhibit tooth root resorption induced by orthodontic force [223]. It is hypothesized that the excessive release of calcium ions from root surface microcracks can induce cellular inflammation and apoptosis, thereby activating a cascade response in odontoclasts, which may be a key factor in the etiology of root resorption. Microcracks at the root surface cause an increase in proteins related to calcium ion pathways [206], leading to an inflammatory response [224], which ultimately results in root resorption. The upstream proteins of the calcium ion pathway, NOX2, CamkII, and ANT, were significantly elevated [224, 225]. The downstream proteins Calcineurin [226], and Aifm1 were elevated [227], while Mapk was reduced [228], ultimately leading to cell apoptosis. Cell apoptosis can relieve the inhibition of osteoclasts and odontoclasts, promoting their formation [229].

Summary of this chapter

This chapter systematically examines the repair mechanisms of microdamage in biological hard tissues, particularly bones and teeth, revealing the differences and commonalities in repair pathways for different types of damage (linear microcracks and diffuse damage) and tissue types (bones and teeth). The key finding is that the repair process within organisms is a complex, multi-level (cellular, molecular, mechanical signals) collaborative process. Bone tissue repair primarily involves targeted bone reconstruction initiated by damage signals, such as apoptosis, carried out by basic multicellular units (BMUs), while diffuse damage relies on different non-targeted mechanisms. The repair of odontoid bone is similar to that of bones, but the repair mechanisms of dentin and enamel are less well-understood. These mechanisms elucidate the synergistic effects of microstructure, mechanical stimuli, and molecular signals, providing a theoretical foundation for understanding tissue integrity maintenance, developing biomimetic materials, and formulating clinical strategies (such as promoting repair and inhibiting abnormal absorption). The imbalance in repair capacity is a critical factor in various pathological conditions.

Discussion

Broadly speaking, the formation and propagation of cracks result from the interaction of material properties, loading conditions, and environmental factors, and can be classified as a major category of material damage behavior. In materials science, it is crucial to analyze the mechanical behavior of materials, investigate the mechanisms of crack initiation and propagation, and identify the patterns of crack development. These insights are vital for designing fracture-resistant structures, which in turn helps to improve the quality and performance of structural products, allowing them to operate safely and efficiently at their full potential. Biological hard tissues not only adhere to the principles of microdamage observed in generalized materials science but also possess inherent repair mechanisms. The study of microdamage in these tissues aims ultimately at predicting, controlling, preventing, and even repairing microdamage.

Some research advancements and technological progress often begin in clinical medical research and gradually extend to dental medicine. Current research on microdamage in biological hard tissues mainly focuses on bone tissue. Bone mechanics and bone biology are integrated to form the field of bone biomechanics, which links bone remodeling with the adaptation of internal structures to load and the elimination of fatigue damage. Bone microdamage is caused by abnormal loading on healthy tissue or normal loading on pathological or diseased tissue [230]. Current research on microdamage primarily focuses on linear microcracks with sharp contours, ranging from tens to hundreds of micrometers in length, and diffuse microdamage consisting of clustered sub-lamellar cracks approximately 1–2 μm or smaller [9]. The research value of cross-hatched microcracks remains to be explored. Moreover, as research progresses, new perspectives have emerged. For instance, two-dimensional finite element modeling of lamellar bone suggests that the typical length of microcracks corresponds to the thickness of individual lamellae in the bone structure. However, these conclusions require further validation [231].

Mechanisms for repairing microcracks and diffuse microdamage are different. The reasons why diffuse damage does not induce osteocyte apoptosis and its repair mechanisms remain unclear and warrant further investigation. Current research indicates that the microstructure directly influences the initiation and progression of microcracks. The presence of microdamage alters the microstructure of biological hard tissues, making it a significant factor in determining the quality of these tissues [139]. There is a dynamic balance between the accumulation and repair of microcracks. Disruption of this balance can lead to structural damage, hard tissue resorption, degradation of mechanical properties, and even fractures. The investigation into the mechanical formation mechanisms and propagation characteristics of microcracks aids in the analysis of bone mechanics and provides valuable insights for exercise load optimization and clinical rehabilitation. Recognized as an integral component of bone quality, microdamage is essential for understanding the overall integrity of bone tissue [110]. Microdamage is closely associated with bone remodeling. Aging, disease, and drug treatments can modify remodeling processes, consequently affecting the accumulation of microdamage and the risk of fractures [87]. Research on the mechanisms involved in the absorption and repair processes triggered by microcracks may uncover key factors that stimulate osteocyte apoptosis and initiate bone reconstruction and repair, thereby maintaining bone mass and strength. Research on aging, diseases, and pharmacological interventions may be clinically applied to prevent and treat age-related conditions. Quantifying microdamage and measuring bone mass will contribute to the prediction and prevention of fractures in patients with osteoporosis [152]. In patients undergoing long-term bisphosphonate therapy, the inhibition of bone remodeling leads to an increased number and volume of microcracks, decreased bone mechanical strength, and a higher risk of fractures [133]. Predicting the critical timepoint for microcrack accumulation would allow for the optimization of bisphosphonate treatment duration, thereby increasing bone volume and reducing perforation, while discontinuing the drug before severe microcrack accumulation occurs [133].Bisphosphonates have been widely used in the treatment of children and adults with OI [232]. The association between bisphosphonates and OI, as well as their relationship with microdamage, remains unclear. The literature on OI and microdamage dates back to over a decade ago, with limited recent research available, suggesting it could be a valuable direction for future studies.

Current reports on microdamage in dental hard tissues primarily focus on post-operative clinical procedures, with limited studies on the impacts of external forces, tissue characteristics, aging, diseases, and drug factors, as well as the mechanisms of absorption and repair. Research in the field of orthodontics is more prevalent, indicating a potential future research direction in dentistry. Dentin and other parts of the tooth must possess sufficient mechanical fracture resistance to function effectively throughout a person's life. Insights into the fracture behavior of dentin structures offer valuable information for the development of dentin adhesive restorative materials and tooth restoration techniques. For example, microcracks or microgaps induced by polymerization shrinkage, cyclic loading, thermal and mechanical fatigue at the bonding interface damage the bonding stability and durability [233]. Microcrack propagation may lead to micro-leakage, bacteria invasion, restoration dislodgment, and catastrophic failure [234]. A new self-healing adhesive containing dimethylaminohexadecyl methacrylate (DMAHDMA) and nanoparticles of amorphous calcium phosphate (NACP) with long-term crack-healing, antibacterial and anti-caries properties is promising for applications in a wide range of dental restorations [235]. Research on dentin microcracks resulting from various root canal preparation techniques in endodontic treatment can inform clinical practices, including the selection of instrument taper, instrument motion, preparation methods, cutting file design, and preparation duration. Predicting microcracks in enamel and bone tissue caused by orthodontic forces can help prevent enamel caries and root resorption. Research focusing on the changes in the root surface microstructure under orthodontic forces, analyzing the micro-stress distribution at the root apex, and exploring the mechanical mechanisms of microcrack formation at the root apex can lead to the development of solutions based on the application of orthodontic forces. Research on the molecular and cellular mechanisms by which microcracks lead to root resorption, providing solutions for the prevention and treatment of root resorption from the perspective of blocking molecular pathways, and providing a theoretical basis for research on related drugs for the prevention and treatment of root resorption, as well as researching potential drugs, is a meaningful topic [97].

The impact of age-related changes on the formation of microcracks remains inconclusive. Some studies indicate that microcracks are unrelated to sex [101], and increase exponentially with age [102]. However, earlier research found no significant correlation between age and microdamage [103]. These differing results may stem from variations in sample collection sites and mechanical loading conditions. Consequently, the relationship between microcracks and age needs further validation through more rigorous experiments. It remains unclear whether aging directly influences the occurrence and development of microcracks, or if the changes in biological hard tissues caused by aging, such as alterations in the microstructure of hard tissues, variations in cell number and activity, and changes in the macroscopic properties of tissues, are responsible for affecting microcracks. There are no related studies reported to date.

Most studies on microcracks use animal models, such as mice, dogs, and pigs. These models may not be ideal substitutes for human bones due to differences in load-bearing mechanisms, growth cycles, and bone turnover rates, which have not been fully established in relation to humans. Some drug tests use the same dosage per unit body weight as in humans, but it remains unclear if this approach is suitable given the differences in bone metabolism between species [110]. In addition, the clinical impact of drug-induced microdamage has yet to be confirmed in humans. Although Wenzel suggest that the damage mechanisms in vivo and ex vivo are similar [4]. Only a few studies have managed to obtain samples from treated patients, a process significantly more challenging than obtaining samples from animals. With the current technology for detecting microcracks, conducting non-destructive research on living animals and humans is difficult. This technology also does not permit real-time monitoring of cellular responses to bone damage. Therefore, there is a need for the development of non-invasive in vivo microdamage assessment techniques in the future.

The correlation between AGEs, osteopontin (OPN), collagen cross-linking, and microdamage has been documented in the literature [236, 237]; however, detailed analyses or further investigations into the nature and implications of these correlations remain lacking. The accumulation of AGEs may induce microdamage, and microdamage, in turn, may promote the accumulation of AGEs [236, 237]. The accumulation of AGEs has been observed in bone with aging [236], diabetes [237], and pharmacological treatments [238]. After ribosylating human tibial cancellous bone in vitro and performing 3D quantification of microdamage using micro-CT, it was found that the accumulation of AGEs in the bone matrix significantly altered the quantity and morphology of microdamage, leading to reduced fracture resistance [7]. Sacher analyzed microdamage and its link to trabecular microarchitecture, finding that the DM2 group exhibited higher AGE pentosidine levels, which may contribute to bone matrix embrittlement, increasing pre-yield damage and accelerating microcrack propagation post-yield [113]. This is probably because AGEs may alter the behavior of osteoblasts and osteoclasts, thereby affecting the biomechanical properties of bones [239]. Meanwhile, higher levels of AGEs are reported in regions with increased amounts of crack-like microdamage [240]. Fatigue-induced changes in bone activate OPN, which plays a key structural role after microdamage occurs [241]. It was reported that OPN-rich interfacial zone may be important in minimizing strain-induced fatigue damage and microcrack propagation in bone and across other mineralized tissues interfaces [242]. Microdamage analysis revealed significantly reduced diffuse damage formation in OPN knockout mice compared to WT controls [243]. It has also been suggested that microdamage levels and morphology may reflect alterations in collagen cross-linking [7]. The material properties of bone, degree of mineralization, and microdamage accumulation are all influenced by collagen cross-link formation. Proper collagen cross-link formation seems to directly affect the physiological mineralization process and microdamage accumulation [244].

Current research primarily focuses on in vitro bone sample experiments and has yet to determine the range of force values that cause microcracks and diffuse microdamage. There is no classification system for the types of microcracks or microdamage that different forces produce. In addition, existing studies lack systematic analysis of the quantity, morphology, and distribution of microdamage and the subsequent bone resorption spaces, which are essential indicators for initiating bone remodeling [245]. Currently, there is still no universal agreement regarding the exact process through which osteocytes detect and interpret mechanical stimuli [172].

Author contributions

Yubo Fan designed the work. Xiaojun Cao and Shengzhao Xiao wrote the main manuscript. Canao Shen prepared Figs. 3, 4. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [12202274], the Interdisciplinary Program of Shanghai Jiao Tong University [YG2022QN049].

Data availability

No data sets were generated or analyzed during the current study.

Declarations

Competing interests

The authors declare no competing interests.