Bone regeneration strategy inspired by the study of calcification behavior in deer antler.

Bone regeneration has attracted much attention from various researchers and inspired numerous strategies for bone formation. In this study, rapid calcification of deer antlers was studied to unravel bone biology by investigating mineral composition, morphology and microstructure. Calcification model was hypothesized and preliminarily established by in vitro experiments. In our model, mineral deposition and phase conversions in the gel matrix were mimicked. Results revealed that mineral metabolism including deposition and phase conversion plays key roles in calcification in vivo, which inspired the bone regeneration strategy with three main components, i.e. enhanced mineral nucleation, mineral ions sources and crystals habits. Rapid mineral metabolism of implant apatite biomaterials was supposed as the critical aspect of bone regeneration. This study will provide a relatively ideal model for peer bone regeneration studies.

Manganese Supplementation in Deer under Balanced Diet Increases Impact Energy and Contents in Minerals of Antler Bone Tissue.

Bone ash, collagen, Ca and P composition, are considered the main factors affecting mechanical properties in bones. However, a series of studies in bone and antler have shown that some trace minerals, such as manganese, may play a role whose importance exceeds what may be expected considering their low content. A previous study showed that a reduction in manganese in antlers during a year of late winter frosts led to generalized antler breakage in Spain, which included a reduction of 30% of cortical thickness, 27% reduction in impact energy, and 10% reduction in work to peak force. Starting for this observation, we experimentally studied the effects of manganese supplementation in adults and yearling (yearlings) red deer under a balanced diet. Subjects were 29 deer of different age classes (adult n = 19, yearlings n = 10) that were divided in a manganese injected group (n = 14) and a control group (n = 15). Antler content in ashes and minerals, intrinsic mechanical properties and cross section structure were examined at 4 points along the antler beam. A one way ANOVA (mean per antler) showed that in yearlings, manganese supplementation only increased its content and that of Fe. However, in adults, Mn supplementation increased the mean content per antler of Ca, Na, P, B, Co, Cu, K, Mn, Ni, Se (while Si content was reduced), and impact work but not Young's modulus of elasticity, bending strength or work to peak force. A GLM series on characteristics in the uppermost part examined in the antler, often showing physiological exhaustion and depletion of body stores, showed also a 16% increase in work to peak force in the antlers of the treated group. Thus, manganese supplementation altered mineral composition of antler and improved structure and some mechanical properties despite animals having a balanced diet.

Histocompositional organization and toughening mechanisms in antler.

Mechanical testing studies by Krauss et al. (2009) and Gupta et al. (2013) suggest that the extraordinary toughness of antler bone is primarily achieved by intrinsic/nanostructural mechanisms instead of extrinsic/microstructural mechanisms. However, this conclusion is based on data from extremely small specimens from one antler loaded only in tension, which impedes discernment of the relative importance of intrinsic vs. extrinsic mechanisms. In the present study we conducted analyses into the microstructural features of antler for details of potential additional microscale toughening characteristics, as suggested by recent mechanical testing studies of bulk specimens. The data are also considered in view of the above-mentioned studies concluding that extrinsic/microstructural toughening mechanisms are less important than nanoscale/intrinsic toughening mechanisms in antler. Mule deer antlers were evaluated using: (1) backscattered electron imaging for micro-mineralization, (2) circularly polarized light for osteonal interfacial complexity and collagen fiber orientation (CFO) heterogeneity, and (3) X-ray 3D micro-computed tomography for osteon/vessel orientation, density, and size. Results showed: (1) hyper-mineralized seams of approximately 3-4 microns thickness within relatively hypermineralized "zones" that course circuitously along osteonal interfaces, (2) highly heterogeneous CFO, including increased oblique-to-transverse CFO near/adjacent to osteon peripheries, and (3) osteons are often highly elongated in 2D. 3D reconstructions show that a considerable percentage of the vascular canals course obliquely with respect to the antler long axis. While results show multiple possible extrinsic-level histological characteristics in antler bone, it remains to be determined if microstructural characteristics become subsidiary to nanostructural characteristics in enhancing toughness during the majority of post-yield behavior of antler bone when loaded in a biologically relevant fashion.

Region-of-interest tomography using filtered backprojection: assessing the practical limits.

There are many cases where it is desirable to reconstruct at high resolution a small volume from a larger sample. Here we describe the outcomes of a reconstruction trial based on real samples aimed at delineating the practical limits to which a small region of interest can be viewed from a large sample. Our approach has been to artificially truncate the sinograms of whole sample scans to simulate region of interest tomography. A simple filtered back projection algorithm has been applied, with the sinograms extended laterally in a simple manner to make up for the truncated portions. The impact of the degree of truncation (from 0% down to 99%), the number of projections used, as well as the position of the region of interest, on the faithfulness of the reconstruction is evaluated for a range of sample types. We have assessed the nature of, and extent to which, artefacts are introduced and the degree to which simple strategies can minimize them to an acceptable level without the need for complex reconstruction algorithms, projection stitching strategies or very large detectors. It is found that for a wide range of objects the effect of truncation on feature detection is negligible and that excellent images can be reconstructed if the number of projections is calculated not on the basis of the number of pixels on the camera, but on the number of pixels that would be required to scan the whole sample at the chosen pixel resolution. This paper demonstrates that in many cases more sophisticated reconstruction strategies are not necessary.

Morphology and ultrastructure of antler velvet hair and body hair from red deer (Cervus elaphus).

We provide a detailed description of the ultrastructure of deer hair fibers. Guard hairs and underhairs from the winter coat of red deer (Cervus elaphus), and antler velvet hairs from the same species were examined. All fibers displayed the typical keratin fiber morphology of overlapping cuticle cells surrounding a core of cortex cells, and often a centrally-located medulla, but there were considerable differences in the diameter, cuticle thickness, and scale pattern, and in the relative amounts of cortex and medulla along individual fibers, and between the different types of fiber. In addition, closer examination of cortex cells using transmission electron microscopy revealed considerable differences in the arrangement of intermediate filaments in the different fiber types. Fine underhairs appeared similar to fine wool fibers from sheep because intermediate filament arrangements were very similar to those found in wool orthocortex cells and paracortex cells. In addition, a similar bilateral distribution of these cell types was evident. However, in the antler velvet hairs and the guard hairs, intermediate filament arrangements were more variable and complex, and showed similarities to those in heterotype cortex cells described for human hair.

Nano-mechanical properties of individual mineralized collagen fibrils from bone tissue.

Mineralized collagen fibrils (MCFs) are distinct building blocks for bone material and perform an important mechanical function. A novel experimental technique using combined atomic force microscopy and scanning electron microscopy is used to manipulate and measure the mechanical properties of individual MCFs from antler, which is a representative bone tissue. The recorded stress-strain response of individual MCFs under tension shows an initial linear deformation region for all fibrils, followed by inhomogeneous deformation above a critical strain. This inhomogeneous deformation is indicative of fibrils exhibiting either yield or strain hardening and suggests possible mineral compositional changes within each fibril. A phenomenological model is used to describe the fibril nano-mechanical behaviour.

[Microscopical identification and hierarchical cluster analysis of seven kinds of pilose antler velvet].

OBJECTIVE: According to the microscopical features of seven kinds of pilose antler velvet, to identify various kinds of pilose antler. METHODS: To identify seven kinds of pilose antler velvet by bio-microscope, then the classification was carried out by hierarchical cluster analysis. RESULTS: The microscopic identification of seven kinds of pilose antler velvet indicators were made and effectively classified. CONCLUSION: This method is useful in the identification of pilose antler,and provides reference for its qualitative evaluation.

Histological studies of growing and mature antlers of red deer stags (Cervus elaphus).

This study aims at presenting histology of growing and mature antlers in red deer stag (Cervus elaphus). Growing antlers constitute a model organ for examining regeneration processes of tissues because they are the only mammalian appendages capable of regeneration. Histological study revealed that the tip of a growing antler consists of hairy skin, perichondrium, mesenchyme and chondroprogenitors area. By performing immunochistochemistry, we found that cell expressing Ki-67 and PCNA antigens were localized in basal layer of epidermis, skin glands and beneath their secretory sections, mesenchyme as well as within and in the vicinity of central blood vessels. Ultrastructurally, cells from chondroprogenitors zone have chondroblast-like morphology and take part in producing of collagen fibres followed by the process of cartilage mineralization. However, mature antlers also consist of lamellar osseous tissue.

Nature's answer to breaching the skin barrier: an innovative development for amputees.

The human body has evolved to maintain homeostasis through the covering of skin and mucous membranes, which separate the internal environment from the harsh and variable external milieu. Few structures naturally penetrate these coverings, and teeth are the only exception in human beings. Attempts to breach these barriers, to develop skin- penetrating, bone-anchored amputation prostheses, can lead to opportunist invasion by microorganisms and subsequent infection, which can jeopardize the life of the individual. There are only a few fascinating examples where the integument of other species is interrupted without such dire consequences, and the deer antler is one such case. Deer antlers are cephalic bony appendages arising from the frontal bones of the skull of the males of most deer species, and are true transient skin-penetrating structures. Antlers are subject to extreme loading during the rutting season and yet the skin-bone barrier remains intact. Here we show how deer antlers can be used as natural analogues for the successful development of specialized orthopaedic amputation prosthetics. We have used quantitative and qualitative findings from a study of the morphology of deer antlers to develop a device that mimics their structure, which creates a tight seal between the implant and the host tissues, for use in amputation prosthetics.

Histological structure of antlers in castrated male fallow deer (Dama dama).

Antlers are periodically replaced cranial appendages that, except for the reindeer, are grown only by male deer. The annual antler cycle is controlled by seasonal fluctuations of sex steroid concentrations in the blood, and accordingly castration of male deer causes deviations from normal antler growth. The present study investigated antler histology of castrated fallow bucks (Dama dama). Castration in early spring was followed by casting of the hard antlers carried by the bucks and the growth of a new set of antlers, which remained in velvet permanently. In the following year, numerous bony protuberances developed from the original antler surface. Further growth of these protuberances, which were formed by subperiosteal intramembranous ossification, led to a marked increase in antler diameter in the affected areas. Compared to antlers of intact bucks, the antlers of the castrates showed histological signs of immaturity, suggestive of a reduced bone remodeling and an impairment of the mineralization process. These changes point to the dependence of the above processes on a stimulation by higher levels of sex steroids. Two years after castration, the antlers also developed integumental thickening and showed an initial formation of skin outgrowths. Cystic structures were present in the skin, which were often filled with a presumably sebaceous and/or keratinous material. Formation of intradermal bone or cartilage was not observed in the antlers of the castrated fallow bucks. The histological structure of the skin outgrowths suggested that they were caused by a hypertrophy of the dermal component of the velvet. Due to the localized bone overgrowth, resulting from the periosteal bone apposition onto the original antler surface, skin-lined infoldings originated, which reached deep into the newly formed bone. Our study revealed no indication of invasive/destructive bone growth in the antlers, i.e., of a penetration of the newly formed bone tissue into the pre-existing bone. The hypertrophic bone growth in the antlers of the castrates is compared with other forms of periosteally derived hypertrophic bone formation, including osteomas, in the mammalian skeleton. It is discussed whether the skin and bone outgrowths of the antlers of castrated fallow bucks may be classified as benign tumors.

Histological studies of bone formation during pedicle restoration and early antler regeneration in roe deer and fallow deer.

The purpose of the present study was to examine the process of bone formation in the regenerating cranial appendages of roe deer (Capreolus capreolus) and fallow deer (Dama dama) during the early postcasting period. After the antlers are cast, osteoclastic and osteoblastic activities lead to a smoothing of the pedicle's separation surface, a strengthening of the pedicle bone, and a partial restoration of the distal pedicle portion that was lost along with the cast antler. Initially, bone formation occurs by intramembranous ossification, but early during the regeneration process cartilage is formed at the tips of the cranial appendages, and is subsequently replaced by bone in a process of endochodral ossification. Shortly after the antlers are cast, the cambium layer of the periosteum in the distal pedicle is markedly enlarged, which suggests that the periosteum serves as a cell source for the bone-forming tissue covering the exposed pedicle bone. The histological findings of our study are consistent with the view that the bony component of the regenerating cranial appendages of deer is largely derived from the pedicle periosteum. Based on findings in other bone systems, we speculate that stem cells that can undergo both osteogenic and chondrogenic differentiation are present in the pedicle periosteum. The early onset of chondrogenesis in the regeneration process is regarded as an adaptation to the necessity of producing a huge volume of bone within a short period. This parallels the situation in other cases of chondrogenesis in membrane bones.

Experimental validation of a microcracking-based toughening mechanism for cortical bone.

It has been proposed that cortical bone derives its toughness by forming microcracks during the process of crack propagation (J. Biomech. 30 (1997) 763; J. Biomech. 33 (2000) 1169). The purpose of this study was to experimentally validate the previously proposed microcrack-based toughening mechanism in cortical bone. Crack initiation and propagation tests were conducted on cortical bone compact tension specimens obtained from the antlers of red deer. For these tests, the main fracture crack was either propagated to a predetermined crack length or was stopped immediately after initiating from the notch. The microcracks produced in both groups of specimens were counted in the same surface area of interest around and below the notch, and crack growth resistance and crack propagation velocity were analyzed. There were more microcracks in the surface area of interest in the propagation than in initiation specimens showing that the formation of microcracks continued after the initiation of a fracture crack. Crack growth resistance increased with crack extension, and crack propagation velocity vs. crack extension curves demonstrated the characteristic jump increase and decrease pattern associated with the formation of microcracks. The scanning electron micrographs of crack initiation and propagation displayed the formation of a frontal process zone and a wake, respectively. These results support the microcrack-based toughening mechanism in cortical bone. Bone toughness is, therefore, determined by its ability to form microcracks during fracture.

Histochemical and ultrastructural studies of cartilage resorption and acid phosphatase activity during antler growth in fallow deer (Dama dama).

Cartilage resorption in forming primary fallow deer antlers was studied by histochemistry and electron microscopy. A high activity of tartrate-resistant acid phosphatase (TRAP), a histochemical marker of skeletal resorbing cells, was first detected in cells located in the mesenchymal tissue separating the columns of hypertrophic cartilage. No cartilage resorption was observed in this region. Intense TRAP staining occurred in large multinucleated cells (identified as inactive osteoclasts) as well as in smaller cells (regarded as mononuclear osteoclast progenitors). On the basis of these findings it was concluded that this was the region where osteoclasts differentiated from progenitor cells. Further proximally, the mineralized cartilage was eroded by active osteoclasts that were located in Howship's lacunae and exhibited an intense TRAP staining. Electron microscopy showed that the cells identified as inactive osteoclasts lacked a polarized organization. In contrast, the active osteoclasts in the zone of cartilage resorption exhibited a typical polarized organization: the nuclei congregated near the basolateral cell surface, and there was a zone of deep membrane infoldings (ruffled border) surrounded by a clear zone at the apical cell pole adjacent to the resorption surface of the mineralized cartilage. The multinucleated cartilage-resorbing cells of the forming antler thus exhibited the typical histochemical and morphological features of active mammalian osteoclasts. Low levels of TRAP activity were also observed in hypertrophic chondrocytes; however, the specificity and potential significance of this staining remain to be elucidated.

Ultrastructural aspects of cartilage formation, mineralization, and degeneration during primary antler growth in fallow deer (Dama dama).

Due to their rapid growth, regular replacement and easy accessibility, deer antlers are considered a useful model for the study of cartilage and bone differentiation and mineralization in mammals. The present study describes, for the first time, the cellular and extracellular matrix changes associated with cartilage formation, mineralization and degeneration in primary antlers on the ultrastructural level. Growing primary antlers of 3 to 4 cm length were obtained from six fallow bucks, aged about 10 months. It was shown that the chondroblasts were derived from progenitor cells of the antler perichondrium and differentiated into mature chondrocytes that subsequently underwent hypertrophic changes. Concomitant with cell hypertrophy, formation of a lacunar and a perilacunar extracellular matrix was observed, the latter containing numerous collagenous fibers. Mineralization of the extracellular matrix occurred via matrix vesicles and the formation of apatite crystals at distinct sites of the collagenous fibers. The hypertrophic chondrocytes of the mineralized cartilage then degenerated, a process that was also occasionally observed in more distally located cells surrounded by still unmineralized matrix. No morphological indications of a transdifferentiation of hypertrophic chondrocytes into bone forming cells, i.e., co-occurrence of a degenerating chondrocyte and a viable osteogenic cell in intact lacunae, were found. The cellular and extracellular matrix changes seen in primary antlers resemble those described for secondary antlers. Our results further indicate that the hypertrophic chondrocytes of primary antlers eventually undergo apoptosis, thereby providing further evidence that metaplastic conversion of cartilage into bone does not play a role in antler growth.

Electron microscopic studies of antlerogenic cells from five developmental stages during pedicle and early antler formation in red deer (Cervus elaphus).

Previous studies using light microscopy have revealed that histogenesis of deer pedicle and antler has four ossification stages. The first of these stages is the development of the permanent pedicle. Initial development of the pedicle is from the cellular layer cells of the antlerogenic periosteum and these cells have been termed initial antlerogenic cells (IACs). Apart from the IACs, it has also been shown that the cellular layer cells of the apical periosteum/perichondrium, the peripheral periosteum of pedicles or antlers, and the marginal periosteum surrounding the pedicles are also capable of either partially or fully generating a pedicle or an antler. Therefore, these cells can all be considered antlerogenic cells and called apical antlerogenic cells (AACs), peripheral antlerogenic cells (PACs), and marginal antlerogenic cells (MACs), respectively. The aim of this study was to examine the ultrastructure of these antlerogenic cells, and to determine whether there were ultrastructural correlates with the changes of these antlerogenic cells and ossification stages. The ultrastructure of each type of antlerogenic cells was systematically examined using transmission electron microscopy, at each stage of pedicle and first antler growth. At the first ossification stage, the IACs were spindle-shaped and inactive. The most obvious feature was the presence of abundant intracellular glycogen. The MACs were similar to the IACs. During the early second stage, most of the AACs changed in appearance from preosteoblasts to prechondroblasts. Much less heterochromatin was found in the AACs than in the IACs. The most striking attribute of the AACs was the existence of intracellular collagen fibers. The MACs showed abnormal dilation of the rough endoplasmic reticulum (RER). During the late second stage, the majority of the AACs were prechondroblasts. AAC nucleoli were clearly discernible and the cisternae of the RER were arranged in parallel. The MACs contained a greater proportion of abnormally-dilated RER. During the third stage, the AACs were all prechondroblasts. The Golgi apparatus in these cells was well developed. Many free ribosomes in rosettes were scattered in the cytoplasm. Most cytoplasm of the majority of the MACs was occupied by abnormally-dilated RER (the lumen of the RER was extremely dilated and appeared electron-lucent). During the fourth stage, the AACs were similar to their counterparts from the third stage, but the boundaries of some AACs were ill-defined. Some MACs were found to be undergoing apoptosis. The PACs were becoming less and less active from distal to proximal along the shaft of the antler. It is a novel finding that antlerogenic cells change in appearance and subcellular content from preosteoblasts to prechondroblasts prior to the transition from intramembranous to endochondral ossification during pedicle formation. Therefore, the differentiation process from antlerogenic cells to chondroblasts is a matter of maturation from prechondroblasts to chondroblasts. The fact that the antlerogenic cells are rich in glycogen makes them more like embryonic cells. The local membrane deficiency of some AACs at the fourth stage and the presence of mature collagen fibrils within the AACs may reflect the unusually high demand for collagen fibrils during the period of rapid antler growth.

Effects of castration on antler growth in fallow deer (Dama dama L.).

Morphology and histological structure of antlers grown after castration (performed on March 25) were studied in six young fallow bucks. In the year after castration, antlerogenesis occurred during the species-specific time span, and the shape of the antlers, which remained permanently in velvet, was normal. During a cold period in December/January, the distal parts of the antlers suffered from frostbite and were subsequently detached. The process of sequestration was similar to that leading to normal antler casting. The sequestration sites were soon covered with skin, but (limited) regrowth of antler tissue from the stumps was not observed before late April/early May, i.e., the time of normal antler regeneration. Simultaneously, growth of knobby protuberances started on the surface of the antlers. Histological analysis of biopsies taken on December 20 in the year after castration revealed that the central parts of the antlers consisted of cancellous lamellar bone with mainly secondary osteons. Peripheral to this, the bone tissue (forming the protuberances) was of a more immature nature and exhibited larger intertrabecular spaces. The outermost layer consisted of woven bone formed by intramembranous ossification from the periosteum and was undergoing active growth and remodeling at the time of biopsy. Thus, bone formation at these sites occurred during a period when no antler growth is observed in normal fallow bucks. The velvet covering the bony protuberances was of normal appearance.

An examination of the micromechanics of failure of bone and antler by acoustic emission tests and Laser Scanning Confocal Microscopy.

Bone fails as a result of damage accumulation in the form of microcracks. Antler is in essence less mineralized bone, but is much tougher than ordinary bone. Failure through damage accumulation also happens in antler, so it is clear that antler's microcracks must be pervasive and that natural selection has produced mechanisms for this material which make it more damage-tolerant. We have examined the development of damage in bone and antler by cyclic loading in three-point bending. We have also conducted acoustic emission studies on these two materials during monotonic tensile loading, and used Laser Scanning Confocal Microscopy (LSCM) to observe microcracks in samples which had been mechanically damaged. We found that diffuse microcracking coincides with the appearance of a macroscopic region of 'yield' in the stress/strain curve of bone and antler; it is related to the local stress (or strain) field that microcracks interact with the structure (particularly in antler). Both acoustic emissions studies and LSCM observations indicated that antler may possess additional modes of stable micromechanical failure compared with ordinary bone.

Wave-length dispersive microprobe analysis of coated samples of bulk tissues.

Hypertrophic scars contain highly pleomorphic cells, including many from the erythrocytic series which have been extravasated. The conventional visual mode of SEM cannot distinguish the cell types with certainty except in the case of typical biconcave disc-shaped erythrocytes. Microprobe elemental analysis might be used to differentiate one type from another on the basis of iron and possibly phosphorus (for nucleated cells). Using coated specimens (gold or gold-palladium) precludes simultaneous visual mode SEM with EDX because of energy line interference with phosphorus and other elements. However, wave-length dispersive analysis offers minimal or no interference, and a coated specimen offers the use of a simultaneous visual mode. We wished to determine if useful elemental data could be obtained from specimens previously prepared only with the purpose of SEM mode studies. Therefore they were not prepared according to contemporary optimal methods. Analysis demonstrates that one group of cells contains 45% or more (dry weight concentration, absolute) iron as opposed to markedly low values in other cell types. Values for phosphorus do not appear essentially different among the cell types except in the case of standard erythrocytes where it is very low. Calcium and sulfur content was also examined. Sulfur might be useful in identifying another cell type in the hypertrophic scar. Using cells and matrix in developing deer antler for control values, the ratio of calcium to phosphorus found in the mineralizing matrix was essentially the predicted value. It is concluded, therefore, that even with a substantially heavy coating of gold, values for the elements tested (Fe, P, Ca, S) are not seriously compromised.

Ultrastructural changes associated with the mineralization of deer antler cartilage.

The maturation and mineralization of deer antler cartilage were investigated ultrastructurally by using enzymatic digestions and subsequent staining with ruthenium red (RR) or phosphotungstic acid (PTA). RR staining of matrix granules was observed in the immature prechondroblastic matrix and became more intense as the cartilage matured into a mineralized tissue. The granules got larger and more numerically dense in the mature matrix. There were matrix granules that coalesced around matrix vesicles or remnants of such in the mineralized zone. These granules were observed after demineralization, and they were RR and acidic PTA-positive (they were not susceptible to hyaluronidase nor trypsin digestion, however). It appears that the granules were modified such that the matrix vesicle formed a centralized nidus for mineralization. The growth of hydroxyapatite crystals along matrix granules (which in this zone may or may not represent proteoglycan monomers) may have caused the coalescence. Microfibrils associated with matrix granules probably represented the hyaluronic acid core of the large proteoglycan complexes because of their susceptibility to hyaluronidase digestion.