Identification of key tissue type for antler regeneration through pedicle periosteum deletion

Epimorphic regeneration is the “holy grail” of regenerative medicine. Research aimed at investigating the various models of epimorphic regeneration is essential if a fundamental understanding of the factors underpinning this process are to be established. Deer antlers are the only mammalian appendages that are subject to an annual cycle of epimorphic regeneration. In our previous studies, we have reported that histogenesis of antler regeneration relies on cells resident within the pedicle periosteum (PP). The present study elaborates this finding by means of functional studies involving the deletion of PP. Four yearling and four 2-year-old stags were selected for total PP deletion or partial PP deletion experiments. Of the animals in the total PP deletion group, one showed no signs of antler regeneration throughout the antler growth season. Two showed substantial and one showed marginal delays in antler regeneration (at 34, 20 and 7 days, respectively) compared with the corresponding sham-operated sides. Histological investigation revealed that the delayed antlers were derived from regenerated PP. Unexpectedly, the regenerative capacity of the antler from the total periosteum-deleted pedicles depended on antler length at surgery. Of the four deer that had partial PP deletion, two regenerated antlers exclusively from the left-over PP on the pedicle shafts in the absence of participation from the pedicle bone proper. The combined results from the PP deletion experiments convincingly demonstrate that the cells of the PP are responsible for antler regeneration. The authors thank the New Zealand Foundation of Research, Science and Technology and Deer Industry New Zealand for funding their research.     

Deer antler – A novel model for studying organ regeneration in mammals

Deer antler is the only mammalian organ that can fully grow back once lost from its pedicle – the base from which it grows. Therefore, antlers probably offer the most pertinent model for studying organ regeneration in mammals. This paper reviews our current understanding of the mechanisms underlying regeneration of antlers, and provides insights into the possible use for human regenerative medicine. Based on the definition, antler renewal belongs to a special type of regeneration termed epimorphic. However, histological examination failed to detect dedifferentiation of any cell type on the pedicle stump and the formation of a blastema, which are hallmark features of classic epimorphic regeneration. Instead, antler regeneration is achieved through the recruitment, proliferation and differentiation of the single cell type in the pedicle periosteum (PP). The PP cells are the direct derivatives of cells resident in the antlerogenic periosteum (AP), a tissue that exists in prepubertal deer calves and can induce ectopic antler formation when transplanted elsewhere on the deer body. Both the AP and PP cells express key embryonic stem cell markers and can be induced to differentiate into multiple cell lineages in vitro and, therefore, they are termed antler stem cells, and antler regeneration is a stem cell-based epimorphic regeneration. Comparisons between the healing process on the stumps from an amputated mouse limb and early regeneration of antlers suggest that the stump of a mouse limb cannot regenerate because of the limited potential of periosteal cells in long bones to proliferate. If we can impart a greater potential of these periosteal cells to proliferate, we might at least be able to partially regenerate limbs lost from humans. Taken together, a greater understanding of the mechanisms that regulate the regeneration of antlers may provide a valuable insight to aid the field of regenerative medicine.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Localization and characterization of STRO-1 cells in the deer pedicle and regenerating antler

The annual regeneration of deer antlers is a unique developmental event in mammals, which as a rule possess only a very limited capacity to regenerate lost appendages. Studying antler regeneration can therefore provide a deeper insight into the mechanisms that prevent limb regeneration in humans and other mammals, and, with regard to medical treatments, may possibly even show ways how to overcome these limitations. Traditionally, antler regeneration has been characterized as a process involving the formation of a blastema from de-differentiated cells. More recently it has, however, been hypothesized that antler regeneration is a stem cell-based process. Thus far, direct evidence for the presence of stem cells in primary or regenerating antlers was lacking. Here we demonstrate the presence of cells positive for the mesenchymal stem cell marker STRO-1 in the chondrogenic growth zone and the perivascular tissue of the cartilaginous zone in primary and regenerating antlers as well as in the pedicle of fallow deer (Dama dama). In addition, cells positive for the stem cell/progenitor cell markers STRO-1, CD133 and CD271 (LNGFR) were isolated from the growth zones of regenerating fallow deer antlers as well as the pedicle periosteum and cultivated for extended periods of time. We found evidence that STRO-1(+) cells isolated from the different locations are able to differentiate in vitro along the osteogenic and adipogenic lineages. Our results support the view that the annual process of antler regeneration might depend on the periodic activation of mesenchymal progenitor cells located in the pedicle periosteum. The findings of the present study indicate that not only limited tissue regeneration, but also extensive appendage regeneration in a postnatal mammal can occur as a stem cell-based process.     

梅花鹿重组Galectin-1的表达及多克隆抗体的制备与鉴定

半乳糖凝集素-1（Galectin-1）是最先被报道的哺乳动物半乳糖凝集素,存在于多种组织和细胞内,参与细胞的粘附、增殖、凋亡和炎症反应等多种生理病理过程,并且与免疫系统的调节和肿瘤的发生发展密切相关。鹿茸是哺乳动物中罕见的能够周期性的脱落和再生的附属器官,作为研究哺乳动物器官再生的新模型受到关注。鹿茸再生是一个基于干细胞的过程,定位于角柄骨膜的干细胞是鹿茸再生的基础,Galectin-1在角柄骨膜细胞（pedicle periosteum cell, PPC）中高度表达,提示其在鹿茸再生中发挥着重要的作用。由于尚没有商用的鹿Galectin-1蛋白及其抗体,为进一步研究Galectin-1在鹿茸再生中的生物学功能,需要制备相应的蛋白和抗体,本实验将梅花鹿Galectin-1基因与pET28a连接,并将重组质粒pET28a-Galectin-1转入大肠杆菌（Escherichia coli）BL21(DE3)中诱导表达。用Ni-NTA Agarose亲和层析纯化融合蛋白,免疫兔子制备多克隆抗体。酶联免疫吸附（enzyme-linked immunosorbent assay, ELISA）法检测抗体效价、Western blot 检测抗体特异性、细胞免疫荧光检测Galectin-1在角柄骨膜细胞中的表达情况。结果表明,本实验成功诱导重组原核表达载体pET28a-Galectin-1在BL21(DE3)中表达,通过Ni纯化获得融合蛋白。ELISA结果显示,抗体效价达到1:64000,Western blot结果表明该抗体特异性良好,细胞免疫荧光显示Galectin-1在PPC全细胞中表达。本实验获得了纯化的鹿Galectin-1蛋白和特异性较好的多克隆抗体,为揭示Galectin-1在鹿茸再生调控中的作用提供了重要的实验材料。     

Antler regeneration: a dependent process of stem tissue primed via interaction with its enveloping skin

Deer antlers are unique mammalian appendages in that each year they are cast and fully regenerate from permanent bony protuberances, called pedicles. In a previous study, we found that there is a difference in the degree of association between pedicle bone and its enveloping skin: tight at the distal third and loose at the proximal two thirds of a pedicle stump. The distal part has been termed the "potentiated" region, and the proximal part the "dormant" region. In the present study, pedicle stumps were artificially created in yearling sika deer by cutting off the tissue distal to either the potentiated or the dormant region. A piece of impermeable membrane was then inserted into the space between the bone and the skin of each treated pedicle stump, while the control pedicles had the same surgery without membrane insertion. The results showed that the inserted membrane blocked pedicle skin participation in the process of antler regeneration. All three potentiated bony pedicle stumps regenerated skin-less antlers; whereas, one of the three dormant bony pedicle stumps failed to regenerate any antler tissue. The other two dormant stumps eventually regenerated normal antlers; however, this only occurred after loss of the inserted membrane. No antler tissue regenerated from the dormant stumps while the inserted membrane remained in place (up to 55 days). All control pedicle stumps regenerated normal antlers. Therefore, we conclude that it is the pedicle bone, but not pedicle skin, that gives rise to regenerating antlers, and that pedicle bone can acquire the potential to regenerate an antler only when it is primed via interaction with its enveloping skin.     

Antler stem cells and their potential in wound healing and bone regeneration

Compared to other vertebrates, the regenerative capacity of appendages in mammals is very limited. Deer antlers are an exception and can fully regenerate annually in postnatal mammals. This process is initiated by the antler stem cells (AnSCs). AnSCs can be divided into three types: (1) Antlerogenic periosteum cells (for initial pedicle and first antler formation); (2) Pedicle periosteum cells (for annual antler regeneration); and (3) Reserve mesenchyme cells (RMCs) (for rapid antler growth). Previous studies have demonstrated that AnSCs express both classic mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs), and are able to differentiate into multiple cell types in vitro. Thus, AnSCs were defined as MSCs, but with partial ESC attributes. Near-perfect generative wound healing can naturally occur in deer, and wound healing can be achieved by the direct injection of AnSCs or topical application of conditioned medium of AnSCs in rats. In addition, in rabbits, the use of both implants with AnSCs and cell-free preparations derived from AnSCs can stimulate osteogenesis and repair defects of bone. A more comprehensive understanding of AnSCs will lay the foundation for developing an effective clinical therapy for wound healing and bone repair.     

Morphological observation of antler regeneration in red deer (Cervus elaphus)

Deer antler offers a unique opportunity to explore how nature solves the problem of mammalian appendage regeneration. Annual antler renewal is an example of epimorphic regeneration, which is known to take place through initial blastema formation. Detailed examination of the early process of antler regeneration, however, has thus far been lacking. Therefore, we conducted morphological observations on antler regeneration from naturally cast and artificially created pedicle/antler stumps. On the naturally cast pedicle stumps, early antler regeneration underwent four distinguishable stages (with the Chinese equivalent names): casting of previous hard antlers (oil lamp bowl), early wound healing (tiger eye), late wound healing and early regeneration (millstone), and formation of main beam and brown tine (small saddle). Overall, no cone-shaped regenerate, a common feature to blastema-based regeneration, was observed. Taken together with the examination on the sagittal plane of each regenerating stage sample, we found that there are considerable overlaps between late-stage wound healing and the establishment of posterior and anterior growth centers. Observation of antler regeneration from the artificially created stumps showed that the regeneration potential of antler remnants was significantly reduced compared with that of pedicle tissue. Interestingly, the distal portion of a pedicle stump had greater regeneration potential than the proximal region, although this differential potential may not be constitutive, but rather caused by whether or not pedicle antlerogenic tissue becomes closely associated with the enveloping skin at the cut plane. Antler formation could take place from the distal peripheral tissues of an antler/pedicle stump, without the obvious participation of the entire central bony portion. Overall, our morphological results do not support the notion that antler regeneration takes place through the initial formation of a blastema; rather, it may be a stem cell-based process.     

Regrowth of amputated velvet antlers with and without innervation

The influence of removing portions of the growing antler of yearling red deer stags on subsequent regeneration of the antler in the same season was studied. The influence of the innervation of the antler on such regeneration was the subject of a further study. When the top 0.5-1 cm was removed from antlers 9-17 cm long, growth was slightly reduced in that season. When the antler/pedicle length was reduced to 6-10 cm in antlers 16-38 cm long, branched antlers regrew in 11 out of 13 cases provided the amputation was carried out early in the growing season, i.e., before mid-December. Denervated antlers were shorter, lighter, and of different shape compared with controls, but they were of similar density. Denervation was confirmed histologically. Cleaning of velvet and casting of antlers following castration were unaffected by denervation. It would appear that although nerves affect the size and shape of the antler, they are not essential to the actual control of antler growth and regeneration.     

Improbable appendages: Deer antler renewal as a unique case of mammalian regeneration

Deer antlers are periodically replaced cranial appendages that develop from permanent outgrowths of the frontal bones known as pedicles. Antler re-growth is a unique regenerative event in mammals which in general are unable to replace bony appendages. Recent evidence suggests that antler regeneration is a stem cell-based process that depends on the activation of stem cells located in the pedicle periosteum which are presumed to be neural crest-derived. It has been demonstrated that several developmental pathways are involved in antler regeneration that are also known to play a role in the control of skeletal development and regeneration in other vertebrates. However, in contrast to most other natural examples of regeneration of complete body structures, antler regeneration apparently neither depends on a functional nerve supply nor involves a direct contact between wound epithelium and mesenchymal tissue. Antlers thus demonstrate that regeneration of a large bony appendage in a mammal can be achieved by a process that differs in certain aspects from epimorphic regeneration in lower vertebrates.     

Deer antler regeneration: cells, concepts, and controversies

The periodic replacement of antlers is an exceptional regenerative process in mammals, which in general are unable to regenerate complete body appendages. Antler regeneration has traditionally been viewed as an epimorphic process closely resembling limb regeneration in urodele amphibians, and the terminology of the latter process has also been applied to antler regeneration. More recent studies, however, showed that, unlike urodele limb regeneration, antler regeneration does not involve cell dedifferentiation and the formation of a blastema from these dedifferentiated cells. Rather, these studies suggest that antler regeneration is a stem-cell-based process that depends on the periodic activation of, presumably neural-crest-derived, periosteal stem cells of the distal pedicle. The evidence for this hypothesis is reviewed and as a result, a new concept of antler regeneration as a process of stem-cell-based epimorphic regeneration is proposed that does not involve cell dedifferentiation or transdifferentiation. Antler regeneration illustrates that extensive appendage regeneration in a postnatal mammal can be achieved by a developmental process that differs in several fundamental aspects from limb regeneration in urodeles.     

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.     

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。     

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.     

Induction of deer antlers by transplanted periosteum. I. Graft size and shape

When discs of frontal periosteum from presumptive antler sites of 6-8 month old male fawns of the fallow deer are grafted beneath the foreleg skin, they will differentiate into pedicle bones and induce small antlers in the overlying integument. These antlers shed their velvet in the fall, and in succeeding years are replaced by larger outgrowths not exceeding 7 cm in length. Periosteal transplants 1.5 cm in diameter gave rise to ectopic antlers in 100% of the grafts, while discs measuring 1.05 cm, 0.75 cm and 0.4 cm did so in only 20% of the cases. Conversely, the donor sites produced antlers in 20-23% of the cases following removal of 1.05 cm or 1.5 cm of periosteum, while 80% and 100% grew antlers after deletions of 0.75 cm and 0.4 cm discs of periosteum, respectively. Semicircular grafts of periosteum induced antler development in most cases, especially when derived from the lateral halves of the antlerogenic region on the frontal bone. These findings confirm that the histogenesis of a deer's first pedicle and antler resides in the frontal periosteum over an area about 1.5 cm wide. They also show that leg skin is capable of antlerogenic development under the inductive influence of frontal periosteum, and that integumental wounding may enhance inductive interactions.     

Pedicle and antler fusion and double-head formation in a sika stag (<Emphasis Type="Italic">Cervus nippon</Emphasis>)

A case of pedicle and antler abnormality in a 12–14 year old sika stag shot in January 2003 in Schleswig-Holstein (Germany) is presented. The abnormality combines fusion of pedicles and antler bases with subsequent double-head formation. Double-heads result from growth of new antler bone without casting of the previous hard antlers. In consequence, two consecutively formed antlers are present in an individual. The stags skull showed a plate-like osseous structure whose broad and slightly elevated central portion was identified as the fused pedicles. The peripheral parts of this osseous plate constituted the second antler growth of the (former) double-head. A cast pair of antlers, which were fused at their bases, had been found in a neighbouring hunting district in the summer of 2002. The close fit between the casting surface of the fused antlers and the surface of the pedicle/antler structure on the stags skull indicated that the antlers had been grown by this stag and were belatedly cast from his fused pedicles. The fused antlers thus constituted the first antler growth of the double-head. We suppose that the broad connection between the fused antlers and the fused pedicles prevented antler casting at the normal time and thereby caused the double-head condition. The presentation of this antler abnormality is taken as an occasion to discuss the significance of pedicles for the normal casting and regeneration of antlers.     

The mechanism of antler casting in the fallow deer.

The process by which antlers are detached from their pedicles was examined histologically in fallow deer castrated in the autumn to induce precocious casting. Osteoclastic erosion across an abscission line between the dead bone of the antler and the living bone of the pedicle was found to be responsible for the separation of the 2. As early as 3 days after castration, osteoclasts and associated lacunae were present on the sides of the pedicle bone. These were then found in progressively deeper locations, by 2 weeks extending across the entire width of the pedicle. Concomitant with the centripetal spread of osteoclasts was the enlargement of Haversian canals, the surfaces of which became lined with osteoclasts. These widening vascular channels within the bone were filled with connective tissue, which in precasting stages formed a mesodermal pad about 1 mm thick. In later stages, a circumferential cleft was excavated beneath the antler burr, and connective tissues from the surrounding pedicle skin invaded the space between the antler and pedicle. After casting, the ingrowing integumental tissues fused with the mesodermal tissues derived from the vascular channels of the pedicle to give rise to an incipient antler bud beneath the scab. The ingrowth of epidermis capable of de novo hair follicle formation gave rise to the future velvet skin that envelops the elongating antler.     

Proteome analysis of red deer antlers

Deer antlers are the only mammalian organs capable of repeated regeneration. Although antlers are known to develop from pedicles, which arise from antlerogenic cells of cranial periosteum, their developmental process is not fully elucidated. For example, while endocrine and environmental factors influence the antler development, it is still unclear which signaling pathways are involved in the transduction of such stimuli. To study the developmental process of antlers and identify proteins functioning in their growth, we have established proteome maps of red deer (Cervus elaphus) antlers. With two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry, we analyzed more than 800 protein spots and identified approximately 130 individual proteins derived from the growing tip of antlers. The overall profile of the antler proteome was dissimilar to those of other types of tissue. Also comparison of proteomes derived from proximal bony tissue and the growing tip of antlers revealed substantial differences. Moreover several cell growth or signaling-related proteins are expressed exclusively in the growing tip, suggesting that these proteins function in the growth and differentiation of antlers. Currently, using the antler proteome maps, we are actively searching for the regulatory factor(s) that may control the antler development.     

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.     

Gene expression of axon growth promoting factors in the deer antler

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deer's normal and pedicle skin.