Regenerative medicine for diseases of the head and neck: principles of in vivo regeneration

The application of endogenous regeneration in regenerative medicine is based on the concept of inducing regeneration of damaged or lost tissues from residual tissues in situ. Therefore, endogenous regeneration is also termed in vivo regeneration as opposed to mechanisms of ex vivo regeneration which are applied, for example, in the field of tissue engineering. The basic science foundation for mechanisms of endogenous regeneration is provided by the field of regenerative biology. The ambitious vision for the application of endogenous regeneration in regenerative medicine is stimulated by investigations in the model organisms of regenerative biology, most notably hydra, planarians and urodeles. These model organisms demonstrate remarkable regenerative capabilities, which appear to be conserved over large phylogenetical stretches with convincing evidence for a homologue origin of an endogenous regenerative capability. Although the elucidation of the molecular and cellular mechanisms of these endogenous regenerative phenomena is still in its beginning, there are indications that these processes have potential to become useful for human benefit. Such indications also exist for particular applications in diseases of the head and neck region. As such epimorphic regeneration without blastema formation may be relevant to regeneration of sensorineural epithelia of the inner ear or the olphactory epithelium. Complex tissue lesions of the head and neck as they occur after trauma or tumor resections may be approached on the basis of relevant mechanisms in epimorphic regeneration with blastema formation.     

Bio-electrosprays: a novel electrified jetting methodology for the safe handling and deployment of primary living organisms

Electrohydrodynamic jetting (EHDJ) which is also known as electrosprays (ES) has recently been elucidated as a unique electrified biotechnique for the safe handling and deployment of living organisms. This high intensity electric field driven jetting methodology is now referred to as "bioelectrosprays" (BES). Previously these charged jets have only been shown to jet-process immortalized cells which have undergone expected cellular behavior when compared with control cells. In this paper we demonstrate the ability to jet process primary living organisms in the stable conejetting mode. Finally the viability of the bio-electrosprayed living organisms has been assessed employing a flow cytometry approach which forms the discussion in this paper. Our findings further establish BES as a competing biotechnique, which could be employed for the deposition of primary living organisms according to a predetermined active cellular architecture. One day this could be used for the fabrication of viable tissues and organs for repair or replacement. These advanced studies carried out on BES have direct widespread applications ranging from developmental biology to regenerative and therapeutic medicine, which are a few amongst several other areas of study within the life sciences.     

Spinal cord regeneration: Lessons for mammals from non‐mammalian vertebrates

Unlike mammals, regenerative model organisms such as amphibians and fish are capable of spinal cord regeneration after injury. Certain key differences between regenerative and nonregenerative organisms have been suggested as involved in promoting this process, such as the capacity for neurogenesis and axonal regeneration, which appear to be facilitated by favorable astroglial, inflammatory and immune responses. These traits provide a regenerative‐permissive environment that the mammalian spinal cord appears to be lacking. Evidence for the regenerative nonpermissive environment in mammals is given by the fact that they possess neural stem/progenitor cells, which transplanted into permissive environments are able to give rise to new neurons, whereas in the nonpermissive spinal cord they are unable to do so. We discuss the traits that are favorable for regeneration, comparing what happens in mammals with each regenerative organism, aiming to describe and identify the key differences that allow regeneration. This comparison should lead us toward finding how to promote regeneration in organisms that are unable to do so. genesis 51:529–544. © 2013 Wiley Periodicals, Inc.     

Understanding positional cues in salamander limb regeneration: implications for optimizing cell-based regenerative therapies

Regenerative medicine has reached the point where we are performing clinical trials with stem-cell-derived cell populations in an effort to treat numerous human pathologies. However, many of these efforts have been challenged by the inability of the engrafted populations to properly integrate into the host environment to make a functional biological unit. It is apparent that we must understand the basic biology of tissue integration in order to apply these principles to the development of regenerative therapies in humans. Studying tissue integration in model organisms, where the process of integration between the newly regenerated tissues and the ‘old’ existing structures can be observed and manipulated, can provide valuable insights. Embryonic and adult cells have a memory of their original position, and this positional information can modify surrounding tissues and drive the formation of new structures. In this Review, we discuss the positional interactions that control the ability of grafted cells to integrate into existing tissues during the process of salamander limb regeneration, and discuss how these insights could explain the integration defects observed in current cell-based regenerative therapies. Additionally, we describe potential molecular tools that can be used to manipulate the positional information in grafted cell populations, and to promote the communication of positional cues in the host environment to facilitate the integration of engrafted cells. Lastly, we explain how studying positional information in current cell-based therapies and in regenerating limbs could provide key insights to improve the integration of cell-based regenerative therapies in the future.KEY WORDS: Integration, Limb regeneration, Positional information, Regenerative medicine, Stem cell     

Partial regeneration of the above-elbow amputated rat forelimb. II. Electrical and mechanical facilitation.

This investigation was undertaken to examine the observations of Becker ('72) pertaining to the electrical facilitation of partial limb regenerative responses by means of Ag-Pt wire couples applied to the limb stumps of young, forelimb-amputated white rats. Additionally, in order to examine the possible role of mechanical effects of such device implantations, we have employed uncoupled devices delivering no current or potential difference. In the present experiments, in response to coupled device implantation, cartilage and bone were actively formed in the vicinity of the Pt electrode tip. These tissues contributed to the lengthwise extension of the limb and to the partial restoration of the distal humeral extremity. In limbs bearing the uncoupled electrical devices, qualitatively similar responses were noted, but osteogenesis was diminished in extent compared to that seen in limbs bearing the active or coupled devices. It is therefore necessary to consider the role of mechanical factors in the elicitation of the observed regenerative responses. Myogenesis was enhanced in electrically stimulated limbs, but not in those rats bearing uncoupled devices.     

Further studies on chromocentres and their implications in regeneration

Previous investigations using a number of invertebrates, as well as regenerative tissues/organs of various vertebrates, have promulgated the hypothesis that heterochromatin, in the form of nuclear chromocentres, is correlated with the ability to regenerate. In order to test the universality of this hypothesis, cells from a variety of additional animals were examined for the presence of nuclear chromocentres. In accordance with the hypothesis, cells from these organisms contained numerous chromocentres. Evidence indicates that chromocentres, double minute chromosomes, chromosome 'dots', and telomeres may be different forms of the same heterochromatin entity.     

Repetitive DNA elements as mediators of genomic change in response to environmental cues

There is no logical or theoretical barrier to the proposition that organismal and cell signaling could transduce environmental signals into specific, beneficial changes in primary structure of noncoding DNA via repetitive element movement or mutation. Repetitive DNA elements, including transposons and microsatellites, are known to influence the structure and expression of protein-coding genes, and to be responsive to environmental signals in some cases. These effects may create fodder for adaptive evolution, at rates exceeding those observed for point mutations. In many cases, the changes are no doubt random, and fitness is increased through simple natural selection. However, some transposons insert at specific sites, and certain regions of the genome exhibit selectively and beneficially high mutation rates in a range of organisms. In multicellular organisms, this could benefit individuals in situations with significant potential for clonal expansion: early life stages or regenerative tissues in animals, and most plant tissues. Transmission of the change to the next generation could occur in plants and, under some circumstances, in animals.     

Three-dimensional culture and bioreactors for cellular therapies

A bioreactor is defined as a specifically designed vessel to facilitate the growth of organisms and cells through application of physical and/or electrical stimulus. When cells with therapeutic potential were first discovered, they were initially cultured and expanded in two-dimensional (2-D) culture vessels such as plates or T-flasks. However, it was soon discovered that bioreactors could be used to expand and maintain cultures more easily and efficiently. Since then, bioreactors have come to be accepted as an indispensable tool to advance cell and tissue culture further. A wide array of bioreactors has been developed to date, and in recent years businesses have started supplying bioreactors commercially. Bioreactors in the research arena range from stirred tank bioreactors for suspension culture to those with various mechanical actuators that can apply different fluidic and mechanical stresses to tissues and three-dimensional (3-D) scaffolds. As regenerative medicine gains more traction in the clinic, bioreactors for use with cellular therapies are being developed and marketed. While many of the simpler bioreactors are fit for purpose, others fail to satisfy the complex requirements of tissues in culture. We have examined the use of different types of bioreactors in regenerative medicine and evaluated the application of bioreactors in the realization of emerging cellular therapies.     

The Manna Effect: A Model of Phytoplankton Patchiness in a Regenerative System

A Monte Carlo model for the growth of phytoplankton in a regenerative system has been constructed. This model assumes that nutrients are regenerated randomly in time and space at the scale which is important to the individual organisms. Under such a regime, organisms originally arrayed in a random fashion assume a patchy distribution within fifteen or twenty divisions, with some species becoming nonrandom in their distribution within five divisions. Other factors, physical or biological, may also induce or enhance patchiness, but normal regenerative processes in a nutrient-limited system are sufficient in themselves to explain the phenomenon. Patchiness should be considered as the normal state for nonmotile organisms.     

Extracellular matrix considerations for scar-free repair and regeneration: Insights from regenerative diversity among vertebrates

The extracellular matrix (ECM) is an essential feature of development, tissue homeostasis and recovery from injury. How the ECM responds dynamically to cellular and soluble components to support the faithful repair of damaged tissues in some animals but leads to the formation of acellular fibrotic scar tissue in others has important clinical implications. Studies in highly regenerative organisms such as the zebrafish and the salamander have revealed a specialist formulation of ECM components that support repair and regeneration, while avoiding scar tissue formation. By comparing a range of different contexts that feature scar-less healing and full regeneration vs. scarring through fibrotic repair, regenerative therapies that incorporate ECM components could be significantly enhanced to improve both regenerative potential and functional outcomes. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Medaka fish stem cells and their applications

Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to...     

BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration

Planarians can be cut into irregularly shaped fragments capable of regenerating new and complete organisms. Such regenerative capacities involve a robust ability to restore bilateral symmetry. We have identified three genes needed for bilaterally asymmetric fragments to regenerate missing body parts. These genes are candidate components of a signaling pathway that controls the dorsal-ventral patterning of many animal embryos: a BMP1/Tolloid-like gene (smedolloid-1), a SMAD4-like gene (smedsmad4-1), and a BMP2/4/DPP-like gene (smedbmp4-1). BMP signaling was involved in the formation of new tissues at the midline of regeneration, the dorsal-ventral patterning of new tissues, and the maintenance of the dorsal-ventral pattern of existing adult tissue in homeostasis. smedbmp4-1 was normally expressed at the dorsal midline. Asymmetric fragments lacking a midline displayed new smedbmp4-1 expression prior to formation of a regenerative outgrowth (blastema). Asymmetric fragments containing the midline displayed expanded smedbmp4-1 expression towards the wound. We suggest injured animals that lack left-right symmetry reset their midline through modulation of BMP activity as an early and necessary event in regeneration.     

Sponge cell aggregation: checkpoints in development indicate a high level of organismal complexity

Studies of regeneration provide insight across many scales of animal biology from the processes of cellular communication to the ecology of whole populations. Sponges are highly regenerative animals, with studies showing adults can both recover large portions of their body after predation or damage due to storms, and even reform whole individuals, via an aggregation stage, from dissociated tissues. While sponges are clearly highly regenerative, few studies actually show dissociated cells forming functional individuals. As sponges often serve as model organisms for studying the development and function of traits in metazoans, determining the universality and mechanics of their regeneration potential is important. We tested the capacity of members of seven sponge species from temperate freshwater and marine environments, from a range of taxonomic positions, and with different habits, to form functional sponges after dissociation. Development to a functional sponge progressed through a series of checkpoints: the sorting of cells and removal of debris; adhesion to a substrate and differentiation of cells; organization of cells into tissues; and regionalization of tissues. Two of the seven species tested, Spongilla lacustris and Haliclona cf. permollis, progressed through all four checkpoints, while the remaining five species progressed to various levels of development before aggregates disintegrated. Our findings highlight three important conclusions: (1) The ability of aggregates to differentiate into functional sponges is not as widespread as previously thought; (2) The species‐specific ability of aggregates to develop to functional sponges appears to be an adaptive trait; and (3) The progression of development in aggregates through checkpoints, which in later development involves formation of tissues and regionalization of tissues, highlights the complexity of the sponge body plan and suggests fundamental rules in development shared across metazoans.     

Regeneration across Metazoan Phylogeny:Lessons from Model Organisms

Comprehending the diversity of the regenerative potential across metazoan phylogeny represents a fundamental challenge in biology.Invertebrates like Hydra and planarians exhibit amazing feats of regeneration,in which an entire organism can be restored from minute body segments.Vertebrates like teleost fish and amphibians can also regrow large sections of the body.While this regenerative capacity is greatly attenuated in mammals,there are portions of major organs that remain regenerative.Regardless of the extent,there are common basic strategies to regeneration,including activation of adult stem cells and proliferation of differentiated cells.Here,we discuss the cellular features and molecular mechanisms that are involved in regeneration in different model organisms,including Hydra,planarians,zebrafish and newts as well as in several mammalian organs.     

Three-dimensional bioprinting in tissue engineering and regenerative medicine

With the advances of stem cell research, development of intelligent biomaterials and three-dimensional biofabrication strategies, highly mimicked tissue or organs can be engineered. Among all the biofabrication approaches, bioprinting based on inkjet printing technology has the promises to deliver and create biomimicked tissue with high throughput, digital control, and the capacity of single cell manipulation. Therefore, this enabling technology has great potential in regenerative medicine and translational applications. The most current advances in organ and tissue bioprinting based on the thermal inkjet printing technology are described in this review, including vasculature, muscle, cartilage, and bone. In addition, the benign side effect of bioprinting to the printed mammalian cells can be utilized for gene or drug delivery, which can be achieved conveniently during precise cell placement for tissue construction. With layer-by-layer assembly, three-dimensional tissues with complex structures can be printed using converted medical images. Therefore, bioprinting based on thermal inkjet is so far the most optimal solution to engineer vascular system to the thick and complex tissues. Collectively, bioprinting has great potential and broad applications in tissue engineering and regenerative medicine. The future advances of bioprinting include the integration of different printing mechanisms to engineer biphasic or triphasic tissues with optimized scaffolds and further understanding of stem cell biology.     

The extracellular microscape governs mesenchymal stem cell fate

Each cell forever interacts with its extracellular matrix (ECM); a stem cell relies on this interaction to guide differentiation. The stiffness, nanotopography, protein composition, stress and strain inherent to any given ECM influences stem cell lineage commitment. This interaction is dynamic, multidimensional and reciprocally evolving through time, and from this concerted exchange the macroscopic tissues that comprise living organisms are formed. Mesenchymal stem cells can give rise to bone, cartilage, tendon and muscle; thus attempts to manipulate their differentiation must heed the physical properties of incredibly complex native microenvironments to realize regenerative goals.     

The future prospects of microbial cellulose in biomedical applications

Microbial cellulose has proven to be a remarkably versatile biomaterial and can be used in wide variety of applied scientific endeavors, such as paper products, electronics, acoustics, and biomedical devices. In fact, biomedical devices recently have gained a significant amount of attention because of an increased interest in tissue-engineered products for both wound care and the regeneration of damaged or diseased organs. Due to its unique nanostructure and properties, microbial cellulose is a natural candidate for numerous medical and tissue-engineered applications. For example, a microbial cellulose membrane has been successfully used as a wound-healing device for severely damaged skin and as a small-diameter blood vessel replacement. The nonwoven ribbons of microbial cellulose microfibrils closely resemble the structure of native extracellular matrices, suggesting that it could function as a scaffold for the production of many tissue-engineered constructs. In addition, microbial cellulose membranes, having a unique nanostructure, could have many other uses in wound healing and regenerative medicine, such as guided tissue regeneration (GTR), periodontal treatments, or as a replacement for dura mater (a membrane that surrounds brain tissue). In effect, microbial cellulose could function as a scaffold material for the regeneration of a wide variety of tissues, showing that it could eventually become an excellent platform technology for medicine. If microbial cellulose can be successfully mass produced, it will eventually become a vital biomaterial and will be used in the creation of a wide variety of medical devices and consumer products.     

Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine

The efficiency of regenerative medicine can be ameliorated by improving the biological performances of stem cells before their transplantation. Several ex-vivo protocols of non-damaging cell hypoxia have been demonstrated to significantly increase survival, proliferation and post-engraftment differentiation potential of stem cells. The best results for priming cultured stem cells against a following, otherwise lethal, ischemic stress have been obtained with brief intermittent episodes of hypoxia, or anoxia, and reoxygenation in accordance with the extraordinary protection afforded by the conventional maneuver of ischemic preconditioning in severely ischemic organs. These protocols of hypoxic preconditioning can be rather easily reproduced in a laboratory; however, more suitable pharmacological interventions inducing stem cell responses similar to those activated in hypoxia are considered among the most promising solutions for future applications in cell therapy. Here we want to offer an up-to-date review of the molecular mechanisms translating hypoxia into beneficial events for regenerative medicine. To this aim the involvement of epigenetic modifications, microRNAs, and oxidative stress, mainly activated by hypoxia inducible factors, will be discussed. Stem cell adaptation to their natural hypoxic microenvironments (niche) in healthy and neoplastic tissues will be also considered.     

Microfluidic devices for studying chemotaxis and electrotaxis

Directed cell migration plays important roles in physiological processes such as host defense, wound healing, cancer metastasis and embryogenesis. Many organisms are capable of directional migration, which can be guided by diverse cellular factors including chemical and electrical cues. Recently, microfluidic devices that consist of small channels with micrometer dimensions are being developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus can be used to study the complex guiding mechanisms for cell migration. In this paper we highlight recent applications of microfluidic devices for cell migration research, with a focus on electric field-directed cell migration, to provide important and timely updates of this rapidly developing research field.     

Regeneration of articular cartilage in healer and non-healer mice

Mammals rarely regenerate their lost or injured tissues into adulthood. MRL/MpJ mouse strain initially identified to heal full-thickness ear wounds now represents a classical example of mammalian wound regeneration since it can heal a spectrum of injuries such as skin and cardiac wounds, nerve injuries and knee articular cartilage lesions. In addition to MRL/MpJ, a few other mouse strains such as LG/J (a parent of MRL/MpJ) and LGXSM-6 (arising from an intercross between LG/J and SM/J mouse strains) have now been recognized to possess regenerative/healing abilities for articular cartilage and ear wound injuries that are similar, if not superior, to MRL/MpJ mice. While some mechanisms underlying regenerative potential have been begun to emerge, a complete set of biological processes and pathways still needs to be elucidated. Using a panel of healer and non-healer mouse strains, our recent work has provided some insights into the genes that could potentially be associated with healing potential. Future mechanistic studies can help seek the Holy Grail of regenerative medicine. This review highlights the regenerative capacity of selected mouse strains for articular cartilage, in particular, and lessons from other body tissues, in general.