Toward a blueprint for regeneration

Tissue regeneration has been studied for hundreds of years, yet remains one of the less understood topics in developmental biology. The recent Keystone Symposium on Mechanisms of Whole Organ Regeneration brought together biologists, clinicians and bioengineers representing an impressive breadth of model systems and perspectives. Members of the growing regeneration community discussed classic and new ideas on mechanisms of regeneration and how these can be applied to regenerative medicine.     

Imaging cardiac extracellular matrices: a blueprint for regeneration

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated.     

Transforming growth factor: beta signaling is essential for limb regeneration in axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-beta). In the present study, the full length sequence of the axolotl TGF-beta1 cDNA was isolated. The spatio-temporal expression pattern of TGF-beta1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-beta signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-beta type I receptor, SB-431542, we show that TGF-beta signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-beta signaling are down-regulated. These data directly implicate TGF-beta signaling in the initiation and control of the regeneration process in axolotls.     

Limb regeneration in vertebrates: regulatory factors

Various regulatory factors are required in epimorphic regeneration of an adult newt limb. These factors (namely, amputational injury, the wound and apical epithelium, nerves (mitogenic agents), hormones (the hormonal milieu), bioelectric fields, probably the immune system, and possibly cyclic nucleotides and heretofore unknown regulators) act in concert and contribute to the developing microenvironment of the regenerate in support of normal regrowth and differentiation.     

Cardiac regeneration in non-mammalian vertebrates

The heart is a robust organ, capable of pumping nutrients and transferring oxygen throughout the body via a network of capillaries, veins and arteries, for the entirety of a human's life. However, the fragility of mammalian hearts is also evident when it becomes damaged and parts of the organ fail to function. This is due to the fact that rather than replenishing the damaged areas with functional cellular mass, fibrotic scar tissue is the preferred replacement, resulting in an organ with functional deficiencies. Due to the mammalian hearts incapability to regenerate following damage and the ever-increasing number of people worldwide suffering from heart disease, tireless efforts are being made to discover ways of inducing a regenerative response in this most important organ. One such avenue of investigation involves studying our distantly related non-mammalian vertebrate cousins, which over the last decade has proved to us that cardiac regeneration is possible. This review will highlight these organisms and provide insights into some of the seminal discoveries made in the heart regeneration field using these amazing chordates.     

Retina of vertebrates: an internal cell reserve for regeneration

The recent data were summarized concerning the presence in the retina of fish, amphibians and birds of additional sources of growth and regeneration, alternative to the already known sources, such as growth zone of eye, pigment epithelium, and cells--precursors of rods, and which are localized in the inner nuclear layer of retina. These sources are represented by as yet not finally identified oval small cells and cells of Muller glia. Both types of cells are capable of proliferating and producing precursors for various differentiated cells, including photoreceptors or their additional precursors. The current immunochemistry data are provided, which were obtained using markers of proliferation, proneural phenotype, and specific cell differentiation in the growing retina and in the retina after various damages. The regulatory mechanisms and methods of the stimulation of proliferation of the cells, which are sources of increase in the number and restoration of photoreceptors, interneurons, and glial cells of vertebrate retina, are discussed.     

Nanocomposite regeneration systems for wound healing

The regeneration system “Biokol” based on the principles of combining synthetic and natural biopolymers has been investigated. The wound dressing consists of “large” (200–250 nm) particles of a synthetic biopolymer and a gel component, which consists of “small” polysaccharide elements 10–20 nm in size. The system can be used both separately and in combination with the gel complex and cell cultures. In properties (vapor and gas permeability, mechanical properties, conductivity, resistance to microbes, etc.), it corresponds to the upper layer of the skin. When applied to the wound, the dressing changes its adhesiveness: first, owing to its hydrophilicity and low hydrophobicity, it closely adheres to the wound surface, and after some time, which corresponds to the time the polysaccharide complex is released from the dressing, it becomes hydrophobic and easily separates from the wound. Owing to these properties, the system can be used at all stages of wound healing.     

Skin wound healing in riboflavin deficiency

Healing of excision and incision wounds was evaluated in riboflavin-deficient rats. The period taken for the epithelialization of excision wounds was 4 to 5 days longer in riboflavin-deficient animals compared to ad libitum-fed or food-restricted weight-matched control groups. Riboflavin deficiency as well as food restriction slowed the rate of wound contraction, the effect of riboflavin deficiency being of greater magnitude. The tensile strength of incision wounds in riboflavin deficiency was reduced to 42% of the ad libitum-fed control and 63% of the weight-matched control values. There was a decrease of 25% in total collagen content of incision wounds, in riboflavin deficiency and its maturity was drastically affected as indicated by a twofold increase in salt solubility (1 M NaCl) and a four-fold increase in the alpha/beta subunit ratio of salt-soluble collagen. Food restriction had similar effects but of lower magnitude. The data suggest that alteration in collagen content and maturity may be responsible for the lower tensile strength of incision wounds in riboflavin-deficient rats. This suggestion was supported by the results of a rehabilitation experiment.     

Specificity of VIIIth nerve regeneration in lower vertebrates.

From the initial studies of Sperry (Am. J. Physiol, 144:735-741, 1945) to more recent investigations, the regenerative capacity of the VIIIth cranial nerve in nonmammalian vertebrates has been noted for its robust and accurate recovery of functional connections after transection. The VIIIth cranial nerve contains nerve fibers that link functionally distinct sensory epithelial to various areas within the central nervous system (CNS), yet after transection these multiple components of the nerve navigate back to their original central target areas, without innervating inappropriate nuclei. A number of factors may be required to establish and direct VIIIth nerve regeneration. Cellular interactions appear to be necessary for the initiation of outgrowth and the maintenance of neural connections. The release of chemotropic substances from target cells has been postulated as the most likely mechanism guiding the reinnervation of central targets. Furthermore, the growth characteristics of these neurons in tissue culture, without target cells present, indicates that intrinsically regulated growth features may also contribute to the process of VIIIth nerve regeneration.     

World Zoo Conservation Strategy: a blueprint for zoo development

People need a sense of purpose, the setting of clear targets, the encouragement and support of others in striving for and achieving tasks that have been set. This is particularly true for zoo personnel, a profession which is often under critical attack, especially from those who have a philosophical difficulty in accepting the idea of animals in captivity. The zoo world, with its huge number of collections and in excess of 600 million annual visitors, represents a grossly under-utilized force for the conservation of endangered species, for the development of scientific knowledge and the increase of public awareness through environmental education programmes. With targets before them, zoos can better provide, maintain and develop their full potential for the good of conservation. The World Zoo Conservation Strategy (WZCS) is not a new beginning, but a codification of the existing activities of responsible zoos, with the purpose of reminding all zoos and their supporters of the Mission Aims, which they should embrace and develop. It forms a set of standards of activity against which zoos can judge themselves and be judged by others. Zoological collections' ability to achieve the obligations of the WZCS will depend on public support. The Strategy, whilst setting the activity priorities, is also one which will evolve to meet the changing demands of the conservation world.     

A phylogenetic blueprint for a modern whale

The emergence of Cetacea in the Paleogene represents one of the most profound macroevolutionary transitions within Mammalia. The move from a terrestrial habitat to a committed aquatic lifestyle engendered wholesale changes in anatomy, physiology, and behavior. The results of this remarkable transformation are extant whales that include the largest, biggest brained, fastest swimming, loudest, deepest diving mammals, some of which can detect prey with a sophisticated echolocation system (Odontoceti – toothed whales), and others that batch feed using racks of baleen (Mysticeti – baleen whales). A broad-scale reconstruction of the evolutionary remodeling that culminated in extant cetaceans has not yet been based on integration of genomic and paleontological information. Here, we first place Cetacea relative to extant mammalian diversity, and assess the distribution of support among molecular datasets for relationships within Artiodactyla (even-toed ungulates, including Cetacea). We then merge trees derived from three large concatenations of molecular and fossil data to yield a composite hypothesis that encompasses many critical events in the evolutionary history of Cetacea. By combining diverse evidence, we infer a phylogenetic blueprint that outlines the stepwise evolutionary development of modern whales. This hypothesis represents a starting point for more detailed, comprehensive phylogenetic reconstructions in the future, and also highlights the synergistic interaction between modern (genomic) and traditional (morphological + paleontological) approaches that ultimately must be exploited to provide a rich understanding of evolutionary history across the entire tree of Life.     

Pit organs in axolotls: a second class of lateral line neuromasts

The lateral line system of axolotls (Ambystoma mexicanum) consists of mechanoreceptive neuromasts and electroreceptive ampullary organs. All neuromasts in salamanders are located superficially and are organized into lines that are homologous to canal neuromasts in fishes. Ampullary organs are confined to the head and generally are located adjacent to the lines of superficial neuromasts. Axolotls, however, also possess a third class of receptors; these form restricted patches on the head and are possibly homologous to the superficial pit organs in fishes. In order to test this hypothesis the morphology of the suspected pit organs was examined with scanning electron microscopy, and a number of their physiological properties were determined. Pit organs are approximately half the size of neuromasts and have fewer hair cells, although these hair cells do possess kinocilia and stereocilia like those of neuromasts. Pit organs also possess cupulae and exhibit a pattern of innervation identical to that of neuromasts. Pit organs and neuromasts also exhibit similar rates of spontaneous activity, are excited by weak water currents but not weak electric stimuli, and are not inhibited by magnesium ions. Pit organs appear to have slightly lower rates of spontaneous discharge than neuromasts, however, and have slightly lower displacement thresholds to low frequency wave stimuli. These data support the contention that the pit organs of axolotls constitute a second class of neuromasts homologous to the pit organs of fishes.     

Pattern-deficient forelimb regeneration in adult bullfrogs

This study was designed to test the ability of adult bullfrogs (Rana catesbeiana) to regenerate forelimbs, both with and without various experimental treatments. Distal humerus-level forelimb amputations provided with additional deviated (sciatic) nerve and/or repeated soft-tissue injury exhibited considerable outgrowth. However, control sham-operated forelimbs also produced regenerates with comparable frequency, size, and morphological complexity. The lengths of the regenerates ranged from 0.4 to 2.6 cm, representing an outgrowth of 10-65% of the portion removed by the distal humerus amputation plane; some regenerates exhibited an external morphology indicative of digitlike structures. Some outgrowths were flexible but only one was capable of independent movement. Victoria Blue staining of whole regenerates revealed a variety of internal cartilage elements. Staining showed a single solid mass of cartilage in some regenerates while others had several individual and variably shaped cartilages projecting distally. Histological analysis also revealed the presence of connective tissue, striated muscle, and abundant nerve fibers in addition to the individual cartilage elements. We have tentatively termed these responses pattern-deficient regeneration.