Differentiation and growth of kype skeletal tissues in anadromous male Atlantic salmon (Salmo salar)

The re-initiation of bone development in adult starving Atlantic salmon (Salmo salar) during their energetically expensive upstream migration is remarkable and deserves closer examination. Dramatic alterations of the skull bones and teeth, most prominently, the development of a kype in males, are widely known but little studied or understood. We describe the microstructure and the cellular processes involved in the formation of the skeletal tissues of the kype. Fresh bone material, obtained from animals migrating upstream was subjected to radiological, histological or histochemical analysis. We show that the kype is, in part, composed of rapidly growing skeletal needles arising at the tip of the dentary. Proximally, the needles anastomose into a spongiosa-like meshwork which retains connective tissue inside bone marrow spaces. Ventrally, the needles blend into Sharpey fiber bone. Skeletal needles and Sharpey fiber bone can be distinguished from the compact bone of the dentary by radiography. Rapid formation of the skeleton of the kype is demonstrated by the presence of numerous osteoblasts, a broad distal osteoid zone, and the appearance of proteoglycans at the growth zone. The mode of bone formation in anadromous males can be described as 'making bone as fast as possible and with as little material as possible'. Unlike the normal compact bone of the dentary, the new skeletal tissue contains chondrocytes and cartilaginous extracellular matrix. Formation of the skeleton of the kype resembles antler development in deer (a form of regeneration), or hyperostotic bone formation in other teleost fishes, rather than periosteal bone growth. The type of boneformation may be understandable in the light of the animals' starvation and the energetic costs of upstream migration. However, the structured and regulated mode of bone formation suggests that the skeleton of the kype has functional relevance and is not a by-product of hormonal alterations or change of habitat.     

A role for retinoic acid in regulating the regeneration of deer antlers

Deer antlers are the only mammalian organs that can be repeatedly regenerated; each year, these complex structures are shed and then regrow to be used for display and fighting. To date, the molecular mechanisms controlling antler regeneration are not well understood. Vitamin A and its derivatives, retinoic acids, play important roles in embryonic skeletal development. Here, we provide several lines of evidence consistent with retinoids playing a functional role in controlling cellular differentiation during bone formation in the regenerating antler. Three receptors (alpha, beta, gamma) for both the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families show distinct patterns of expression in the growing antler tip, the site of endochondral ossification. RAR alpha and RXR beta are expressed in skin ("velvet") and the underlying perichondrium. In cartilage, which is vascularised, RXR beta is specifically expressed in chondrocytes, which express type II collagen, and RAR alpha in perivascular cells, which also express type I collagen, a marker of the osteoblast phenotype. High-performance liquid chromatography analysis shows significant amounts of Vitamin A (retinol) in antler tissues at all stages of differentiation. The metabolites all-trans-RA and 4-oxo-RA are found in skin, perichondrium, cartilage, bone, and periosteum. The RXR ligand, 9-cis-RA, is found in perichondrium, mineralised cartilage, and bone. To further define sites of RA synthesis in antler, we immunolocalised retinaldehyde dehydrogenase type 2 (RALDH-2), a major retinoic acid-generating enzyme. RALDH-2 is expressed in the skin and perichondrium and in perivascular cells in cartilage, although chondroprogenitors and chondrocytes express very low levels. At sites of bone formation, differentiated osteoblasts which express the bone-specific protein osteocalcin express high levels of RALDH2. The effect of RA on antler cell differentiation was studied in vitro; all-trans-RA inhibits expression of the chondrocyte phenotype, an effect that is blocked by addition of the RAR antagonist Ro41-5253. In monolayer cultures of mesenchymal progenitor cells, all-trans-RA increases the expression of alkaline phosphatase, a marker of the osteoblastic phenotype. In summary, this study has shown that antler tissues contain endogenous retinoids, including 9-cis RA, and the enzyme RALDH2 that generates RA. Sites of RA synthesis in antler correspond closely with the localisation of cells which express receptors for these ligands and which respond to the effects of RA.     

Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice.

Fracture repair recapitulates in adult organisms the sequence of cell biological events of endochondral ossification during skeletal development and growth. After initial inflammation and deposition of granulation tissue, a cartilaginous callus is formed which, subsequently, is remodeled into bone. In part, bone formation is influenced also by the properties of the extracellular matrix of the cartilaginous callus. Deletion of individual macromolecular components can alter extracellular matrix suprastructures, and hence stability and organization of mesenchymal tissues. Here, we took advantage of the collagen IX knockout mouse model to better understand the role of this collagen for organization, differentiation and maturation of a cartilaginous template during formation of new bone. Although a seemingly crucial component of cartilage fibrils is missing, collagen IX-deficient mice develop normally, but are predisposed to premature joint cartilage degeneration. However, we show here that lack of collagen IX alters the time course of callus differentiation during bone fracture healing. The maturation of cartilage matrix was delayed in collagen IX-deficient mice calli as judged by collagen X expression during the repair phase and the total amount of cartilage matrix was reduced. Entering the remodeling phase of fracture healing, Col9a1(-/-) calli retained a larger percentage of cartilage matrix than in wild type indicating also a delayed formation of new bone. We concluded that endochondral bone formation can occur in collagen IX knockout mice but is impaired under conditions of stress, such as the repair of an unfixed fractured long bone.     

Tissue interactions and antlerogenesis: new findings revealed by a xenograft approach.

Tissue interactions play a pivotal role in organogenesis. Here we describe a xenograft approach to investigate how heterotypic tissue interactions control antler formation in deer. Deciduous antlers grow from the apices of permanent protuberances, called pedicles. Histogenesis of pedicles depends on the antlerogenic periosteum (AP). Pedicles and growing antlers are made up of interior osseocartilage (a mixture of bone and cartilaginous tissue) and exterior skin. In a previous study we hypothesised that pedicle growth may result from mechanical interactions between the interior and exterior components whereas antler generation from a pedicle would involve molecules communicating between the interior and exterior components. To test this hypothesis, we subcutaneously transplanted AP of red deer (Cervus elaphus), either alone or with future pedicle skin, onto nude mice. The results showed that under the nude mouse skin, subcutaneously xenografted AP alone not only could form pedicle-shaped protuberances but also could differentiate into well-organised pedicle-like structures. The overlying mouse skin accommodated the expansion of the grafted AP by initial mechanical stretching and subsequent formation of new skin. Nude mouse skin was not capable of participating in antler tissue formation. However, grafted deer skin together with AP may have successfully rescued this failure after wounding, which highlights the necessity of the specificity of the overlying skin for antler tissue generation. Therefore, we conclude that it is the interaction between the antlerogenic tissue and the overlying skin that results in antlerogenesis: reciprocal mechanical interactions cause pedicle formation, whereas reciprocal instructive interactions induce first antler generation.     

Tissue collection methods for antler research

The rapid growth of deer antlers makes them potentially excellent models for studying tissue regeneration. In order to facilitate this, we have developed and refined antler tissue sampling methods through years of antler research. In the study, antler tissues were divided into three main groups: antler stem tissue, antler blastema and antler growth centre. For sampling stem tissue, entire initial antlerogenic periosteum (around 22 mm in diameter) could be readily peeled off from the underlying bone using a pair of rat-toothed forceps after delineating the boundary. Apical and peripheral periosteum/ perichondrium of pedicle and antler could only be peeled off intact when they were cut into 4 quadrants and 0.5 cm-wide strips respectively. Antler blastema included blastema per se, and potentiated and dormant periostea. Blastema per se was sampled after it was divided into 4 quadrants using a disposable microtome blade. Potentiated and dormant periostea were collected following the same method used for sampling peripheral periosteum of pedicle and antler. The antler growth centre was divided with a scalpel into 5 layers according to distinctive morphological markers. The apical skin layer could be further separated into dermis and epidermis using enzyme digestion for the study of tissue interaction. We believe that the application of modern techniques coupled with the tissue collection methods reported here will greatly facilitate the establishment of these valuable models.     

Role of heterotypic tissue interactions in deer pedicle and first antler formation-revealed via a membrane insertion approach

Heterotypic tissue interactions play an indispensable role in organ generation and regeneration. In contrast to the classic examples of tissue interactions prevailing in the formation of tetrapod limbs or pectoral fins that can only take place when the interactive tissues are in intimate contacts, the interactions in deer antler formation are novel in that the inducer and the responder are separated by a distance of 1-2 mm. This feature offers a unique opportunity to explore the mechanism underlying tissue interactions by permitting membrane insertion between the two interactive tissues. Four experiments were conducted in this study. The results showed that the impermeable membranes inhibited antler formation. In contrast, the permeable membrane (0.45 microm in pore size) substantially slowed pedicle growth and antler initiation but did not stop them. Interestingly, the impermeable membrane/sheath only slightly retarded antler elongation. Overall, our results demonstrate that interactions between the two interactive tissues, antlerogenic tissue and the overlying skin, are indispensable for first antler initiation and are achieved through diffusible molecules rather than direct physical contact. As the heterotypic tissue interactions are only required during antler initiation but not elongation, they must be transient in nature, and thus differ from those operating in limb/fin formation that can only be sustained by continuous interactions. A system in which organ development is achieved only through transient tissue interactions must be novel, if not completely unique. Understanding this system will undoubtedly enrich the knowledge in the field of tissue interactions and organogenesis.     

Antler development and coupled osteoporosis in the skeleton of red deer Cervus elaphus: expression dynamics for regulatory and effector genes

Antlers of deer display the fastest and most robust bone development in the animal kingdom. Deposition of the minerals in the cartilage preceding ossification is a specific feature of the developing antler. We have cloned 28 genes which are upregulated in the cartilaginous section (called mineralized cartilage) of the developing (“velvet”) antler of red deer stags, compared to their levels in the fetal cartilage. Fifteen of these genes were further characterized by their expression pattern along the tissue zones (i.e., antler mesenchyme, precartilage, cartilage, bone), and by in situ hybridization of the gene activities at the cellular level. Expression dynamics of genes col1A1, col1A2, col3A1, ibsp, mgp, sparc, runx2, and osteocalcin were monitored and compared in the ossified part of the velvet antler and in the skeleton (in ribs and vertebrae). Expression levels of these genes in the ossified part of the velvet antler exceeded the skeletal levels 10–30-fold or more. Gene expression and comparative sequence analyses of cDNAs and the cognate 5′ cis-regulatory regions in deer, cattle, and human suggested that the genes runx2 and osx have a master regulatory role. GC–MS metabolite analyses of glucose, phosphate, ethanolamine-phosphate, and hydroxyproline utilizations confirmed the high activity of mineralization genes in governing the flow of the minerals from the skeleton to the antler bone. Gene expression patterns and quantitative metabolite data for the robust bone development in the antler are discussed in an integrated manner. We also discuss the potential implication of our findings on the deer genes in human osteoporosis research.     

The organization of the osteocyte network mirrors the extracellular matrix orientation in bone

Bone is a dynamic tissue that is continually undergoing a process of remodeling - an effect due to the interplay between bone resorption by osteoclasts and bone formation by osteoblasts. When new bone is deposited, some of the osteoblasts are embedded in the mineralizing collagen matrix and differentiate to osteocytes, forming a dense network throughout the whole bone tissue. Here, we investigate the extent to which the organization of the osteocyte network controls the collagen matrix arrangement found in various bone tissues. Several tissue types from equine, ovine and murine bone have been examined using confocal laser scanning microscopy as well as polarized light microscopy and back-scattered electron imaging. From comparing the spatial arrangements of unorganized and organized bone, we propose that the formation of a highly oriented collagen matrix requires an alignment of osteoblasts whereby a substrate layer provides a surface such that osteoblasts can align and, collectively, build new matrix. Without such a substrate, osteoblasts act isolated and only form matrices without long range order. Hence, we conclude that osteoblasts synthesize and utilize scaffold-like primary tissue as a guide for the deposition of highly ordered and mechanically competent bone tissue by a collective action of many cells.     

Antler stiffness in moose (Alces alces): Correlated evolution of bone function and material properties?

The material properties of bone can vary considerably among skeletal elements from different parts of the body that serve different functions. However, functional demands placed on a specific type of skeletal element also can vary at a variety of scales, such as between different parts of the element, among individuals of a species, and across species. Variation in bone material properties might be correlated with differing functional demands at any of these scales. In this study we performed three-point bending tests on bone specimens extracted from antlers of moose (Alces alces) to test for three types of variation in bone material stiffness (Young's modulus): within the antler structure, between populations of moose, and between moose and other deer species. Because superficial portions of the antler are exposed to greater bending stress and strain than deeper portions, and because the antler beam (the basal shaft that attaches to the skull) is subjected to greater bending moments than more distal parts of the antler, we predicted that superficial bone and bone from the beam would be stiffer than bone from other parts of the antler. Instead, we identified no significant differences in these comparisons. There were also no significant differences in antler stiffness between moose from Michigan and the Yukon, even though the rapid growth required of antlers from northern latitudes like the Yukon has the potential to compromise bone material properties. However, moose have significantly stiffer antlers (11.6 +/- 0.45 GPa, mean +/- SE) than any other deer in the odocoileine lineage. Moreover, phylogenetic reconstructions of the evolution of antler stiffness in deer indicate a strong potential that high antler stiffness is a derived feature of moose. The unusual palmate shape of moose antlers likely subjects their antler beams to higher bending moments than found in other odocoileines, a factor that may have contributed to the evolutionary divergence of moose antler stiffness from that of other members of this clade. Although similarities in the mineral composition of bone across species likely limit the overall range of phylogenetic variation in bone material properties, our results demonstrate that evolutionary diversity in bone material properties can show correspondence with phylogenetic differences in mechanical or ecological demands on skeletal elements.     

基于组学技术的鹿茸生物学研究进展

鹿茸是唯一可周期性再生的哺乳动物器官,由软骨、骨、血管、神经及皮肤组织构成。鹿茸再生过程是基于干细胞的增殖和分化,且生长速度极快而不发生癌变。其不仅可作为一种肢体再生的生物医学模型,而且也作为一种研究骨组织生长发育的模型。现代组学技术快速发展,已普遍应用于生物学的各种领域。利用组学技术,在转录和蛋白质水平上,有力地推动了在分子水平上研究鹿茸生物学的进程。本综述拟对组学技术在鹿茸生物学研究中的应用进行总结回顾,并对未来的发展趋势做进一步展望,为鹿茸生物学的深入研究提供参考。     

Distribution of EGF and its receptor in growing red deer antler

Autografts of the osteogenic part of early antler buds placed elsewhere on the skull have been shown by others to give rise to an antler at the site of grafting. This antler becomes covered in velvet skin, is shed at the end of the growing season and will regrow the following year. Thus, it can be concluded that the nature of antler velvet skin is primarily determined by the underlying osteogenic antler tissue to which it is attached. We hypothesise that a paracrine mechanism operates here and is central to communication between the antler osseous compartment and the integument. A signalling system comprising epidermal growth factor (EGF) and its receptor (EGFR) is known to be expressed in osteogenic cells and to play an important role in skin development and growth. This system may therefore play a significant role in determining the nature and speed of growth of velvet skin via paracrine signalling from osteogenic tissue. We have used bright-field microscope immunohistochemistry to determine the distribution of EGF and its receptor in developing red deer antler osseous compartment and integument. EGF was localized throughout the epidermis and epidermal appendages, in cells of the mesenchyme, in chondrocytes, and in cells of the osteoblastic lineage, including osteoprogenitor cells, osteoblasts and osteocytes. There was strong evidence supporting nuclear and nucleolar staining in sebaceous glands and in keratinocytes. The EGFR was similarly expressed in mesenchyme, chondrocytes and osteoblasts. In skin, the distribution of the EGFR was more localized, being expressed strongly in the deeper cells of the epidermis but not in superficial layers, and was absent from nuclei of cells of the epidermis and its appendages. We conclude that this signalling system is widely distributed in growing antler in a manner which suggests it is predominantly autocrine. No clear-cut evidence for paracrine signalling pathways for this system in either integument or osseous compartments was found. The pattern of distribution of the EGFR in the integument was similar to that seen by others in adult human skin. By contrast, in developing antler osseocartilage, the patterns of distribution were similar to those seen in rodent fetal bone. We conclude that antler consists of rapidly growing fetal osseocartilage overlayed by mature velvet.     

Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.     

Antler development was inhibited or stimulated by cryosurgery to periosteum or skin in a central antlerogenic region respectively

Antler development is triggered by interactions between antler stem cells resident in the antlerogenic periosteum (AP) and the niche cells in the upper portion of overlying skin mediated by diffusible molecules. These interactive cell populations are interposed by the lower portion of the skin and the subcutaneous loose connective tissue (SLCT). It is known that mechanical deletion of just the central AP (having an area equivalent to the size of a pedicle base) by cutting through the skin and SLCT effectively stimulates the marginal AP to initiate antler development. This study was designed to investigate whether the SLCT layer plays a role in antler development by acting as a physical barrier. The results showed that the marginal AP failed to give rise to an antler after the central AP was cryosurgically destroyed with the preservation of the collagen structure of the SLCT. Furthermore, antler development was significantly advanced when the collagen structures of the skin and SLCT layers were substantially attenuated by repeated sprays with liquid nitrogen while keeping the central AP intact. Therefore, we conclude that the interposing SLCT layer acts as a physical barrier between antler stem cells and the niche cell types, and that timing of antler development is primarily controlled by the permeability of the SLCT layer to the putative interactive diffusible molecules.     

Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix

ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.     

Foetal Stages of Antler Development

Foetuses of reindeer, Rangifer tarandus tarandus L., were collected at slaughter and studied for structural primordial stages of pedicle formation and antler growth. Fresh foetuses studied in January and February exhibited a round, pale area with an epidermal infolding or groove at the site of the future antler development. Also, in an ethanol-fixed caribou foetus from Alaska, an epidermal invagination could be seen in the area of later pedicle formation. No protruding bone or cartilage was observed as primordial stages of antler growth in reindeer foetuses collected from 10 November to 26 April. It is concluded that an epidermal infolding exists in the foetus of telemetacarpal cervids such as reindeer and caribou.     

Effects of insulin-like growth factor 1 and testosterone on the proliferation of antlerogenic cells in vitro.

Androgen hormones and growth factors are implicated in pedicle formation and antler transformation in deer. The potential to form a pedicle and an antler is only found in the antlerogenic periosteum (AP) overlying the presumptive antler growth region. Histological studies (Li and Suttie, '94) showed that AP consists of an inner cellular layer and an outer fibrous layer. Pedicle and antler are mainly derived from the cellular layer cells of the AP. Ossification takes place in four stages: intramembranous (IMO), transitional (OPC), pedicle endochondral (pECO) and antler endochondral (aECO). However, the precise mechanism whereby androgen hormones and growth factors control pedicle and antler formation is unknown. The aim of this study was to use cell culture techniques to investigate how testosterone and IGF1 affects the proliferation of antlerogenic cells from the four ossification stages of pedicle/antler in vitro. The results showed that in serum-free medium IGF1 stimulated the proliferation of antlerogenic cells from all four ossification stages in a dose-dependent manner. In contrast, testosterone alone did not show any mitogenic effects on these antlerogenic cells. However, in the presence of IGF1, testosterone increased proliferation of the antlerogenic cells from the IMO and the OPC stages (pedicle tissue), and reduced proliferation of the antlerogenic cells from transformation point (TP) and aECO stages (antler tissue). Therefore, the results from the present in vitro study support the in vivo findings that androgen hormones stimulate pedicle formation but inhibit antler growth. The change in the mitogenic effects of testosterone on antlerogenic cells from positive to negative occurs approximately at the change in ossification type from OPC to pECO. Therefore, these results reinforce the hypothesis that the transformation from a pedicle to an antler takes place at the time when the ossification type changes from OPC to pECO rather than at the time when the pedicle grows to its full species-specific height.     

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions’ orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young’s modulus \(E = 1.90\) GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young’s modulus in the preferential direction of 9–16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.     

Induction of antler growth in a congenitally polled Scottish red deer stag.

A congenitally polled red deer stag was captured from a Scottish deer forest and kept in an enclosure for observations. The animal had rudimentary antler pedicles but no antlers, and during five years of study no significant antler development occurred. Amputation of the apex of one antler pedicle in May 1974 when the stat was 12 years of age resulted in the growth of a complete antler on the operated side, and this antler was subsequently cleaned and cast in the normal way and a new antler cycle was initiated. The result illustrates that the primary abnormality in this polled stag lay not in his inability to grow antler, but in his inability to develop fully formed antler pedicles from which normal antler tissue could differentiate. Traumatizing the rudimentary pedicle had the effect of stimulating growth of antler tissue, and once this was formed the process of cleaning, casting and regrowth occurred spontaneously. The incomplete development of the antler pedicles is considered to be responsible for the absence of antlers in the majority of "hummels" in Scotland, and the etiology of the condition is discussed.     

The role of small leucine-rich proteoglycans in collagen fibrillogenesis

Small leucine-rich proteoglycans/proteins (SLRPs) are associated with collagen fibril formation, and therefore important for the proper formation of extracellular matrices. SLRPs are differentially expressed in tissues and during pathological conditions, contributing to the development of connective tissue properties. The binding of SLRPs to collagens have recently been characterized, and may give some clues to the significance of these interactions. In this mini review, we summarize published work in this field, and propose several mechanisms for how SLRPs can control collagen matrix structure and function. SLRPs appear to influence collagen cross-linking patterns. We also propose that the SLRP-collagen interactions can assist in the process of juxtaposing the collagen monomers by steric hindrance or by directly connecting two collagen monomers during the fibril growth.     

Probabilistic failure analysis of bone using a finite element model of mineral–collagen composites

Microdamage accumulation is a major pathway for energy dissipation during the post-yield deformation of bone. In this study, a two-dimensional probabilistic finite element model of a mineral–collagen composite was developed to investigate the influence of the tissue and ultrastructural properties of bone on the evolution of microdamage from an initial defect in tension. The probabilistic failure analyses indicated that the microdamage progression would be along the plane of the initial defect when the debonding at mineral–collagen interfaces was either absent or limited in the vicinity of the defect. In this case, the formation of a linear microcrack would be facilitated. However, the microdamage progression would be scattered away from the initial defect plane if interfacial debonding takes place at a large scale. This would suggest the possible formation of diffuse damage. In addition to interfacial debonding, the sensitivity analyses indicated that the microdamage progression was also dependent on the other material and ultrastructural properties of bone. The intensity of stress concentration accompanied with microdamage progression was more sensitive to the elastic modulus of the mineral phase and the nonlinearity of the collagen phase, whereas the scattering of failure location was largely dependent on the mineral to collagen ratio and the nonlinearity of the collagen phase. The findings of this study may help understanding the post-yield behavior of bone at the ultrastructural level and shed light on the underlying mechanism of bone fractures.