Cell- and tissue-based approaches for cartilage repair

Damage and degeneration of articular joints is a major healthcare concern, due to the association of joint disease with ageing, the current strong demographic changes in the proportion of elderly in the population, and the increased incidence of trauma in a sports-active younger population. These joints are biomechanical organs that transmit load between bones in our skeleton, and the articular cartilage forms a load-bearing surface that covers the bone within the joints. All the forces across the joints are thus transmitted through the cartilage, and it therefore makes an important biomechanical contribution to joint function. The cartilage is particularly prone to damage, and has limited capacity for natural repair. Although joint replacement is successful, it is less so in younger patients. For these patients, there is currently great interest in developing cell-based treatments for the biological repair of articular cartilage.     

Cell- and peptide-based immunotherapeutic approaches for glioma

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor. Although considerable progress has been made in surgical and radiation treatment for glioma patients, the impact of these advances on clinical outcome has been disappointing. Therefore, the development of novel therapeutic approaches is essential. Recent reports demonstrate that systemic immunotherapy using dendritic cells (DCs) or peptide vaccines is capable of inducing an antiglioma response. These approaches successfully induce an antitumor immune response and prolong survival in patients with glioma without major side effects. There are several types of glioma, so to achieve effective therapy, it might be necessary to evaluate the molecular genetic abnormalities in individual patient tumors and design novel immunotherapeutic strategies based on the pharmacogenomic findings. Here, we review recent advances in DC- and peptide-based immunotherapy approaches for patients with gliomas.     

Biomimetic approaches to tendon repair

The linear organization of collagen fibers in tendons results in optimal stiffness and strength at low strains under tensile load. However, this organization makes repairing ruptured or lacerated tendons extremely difficult. Current suturing techniques to join split ends of tendons, while providing sufficient mechanical strength to prevent gapping, are inadequate to carry normal loads. Immobilization protocols necessary to restore tendon congruity result in scar formation at the repair site and peripheral adhesions that limit excursion. These problems are reviewed to emphasize the need for novel approaches to tendon repair, one of which is the development of biomimetic tendons. The objective of the empirical work described here was to produce biologically-based, biocompatible tendon replacements with appropriate mechanical properties to enable immediate mobilization following surgical repair. Nor-dihydroguaiaretic acid (NDGA), a di-catechol from creosote bush, caused a dose dependent increase in the material properties of reconstituted collagen fibers, achieving a 100-fold increase in strength and stiffness over untreated fibers. The maximum tensile strength of the optimized NDGA treated fibers averaged 90 MPa; the elastic modulus of these fibers averaged 580 MPa. These properties were independent of strain rates ranging from 0.60 to 600 mm/min. Fatigue tests established that neither strength nor stiffness were affected after 80 k cycles at 5% strain. Treated fibers were not cytotoxic to tendon fibroblasts. Fibroblasts attached and proliferated on NDGA treated collagen normally. NDGA-fibers did not elicit a foreign body response nor did they stimulate an immune reaction during six weeks in vivo. The fibers survived 6 weeks with little evidence of fragmentation or degradation. The polymerization scheme described here produces a fiber-reinforced NDGA-polymer with mechanical properties approaching an elastic solid. The strength, stiffness and fatigue properties of the NDGA-treated fibers are comparable to those of tendon. These fibers are biocompatible with tendon fibroblasts and elicit little rejection or antigenic response in vivo. These results indicate that NDGA polymerization may provide a viable approach for producing collagenous materials that can be used to bridge gaps in ruptured or lacerated tendons. The tendon-like properties of the NDGA-fiber would allow early mobilization after surgical repair. We predict that timely loading of parted tendons joined by this novel biomaterial will enhance mechanically driven production of neo-tendon by the colonizing fibroblasts and result in superior repair and rapid return to normal properties.     

Genomic approaches to DNA repair and mutagenesis

DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways.     

Physiology and pathophysiology of inner ear melanin

The presence of melanin in the inner ear was established more than a century ago, but the exact biological function of the pigment in the labyrinth has yet to be determined. In this brief review, the correlation of pigmentation and inner ear function, as well as the presumed role of melanocytes in hereditary diseases are discussed. Special attention was drawn to the composition of melanin and its presumed function as a biological reservoir for divalent ions and as an ion exchanger, as well as an intracellular buffering system for calcium. It is pointed out that melanin is capable of binding ototoxic drugs. Finally, morphological responses of melanocytes to local disturbance of Ca++ homeostasis in the inner ear are described as 1) intracellular movement and intraepithelial deposition of melanosomes; 2) cell motility; 3) neomelanogenesis; and 4) enhanced exocytotoxic/endocytotic activity. The possible consequences of this malfunction of the melanocytes on the inner ear function are discussed.     

Ontogeny and regeneration in the inner ear

Brain Cell Biology -     

Distinct contributions from the hindbrain and mesenchyme to inner ear morphogenesis

A mature inner ear is a complex structure consisting of vestibular and auditory components. Microsurgical ablations, rotations, and translocations were performed in ovo to identify the tissues that control inner ear morphogenesis. We show that mesenchyme/ectoderm adjacent to the developing ear specifically governs the shape of vestibular components - the semicircular canals and ampullae - by conferring anteroposterior axial information to these structures. In contrast, removal of individual hindbrain rhombomeres adjacent to the developing ear preferentially affects the growth and morphogenesis of the auditory subdivision, the cochlear duct, or basilar papilla. Removal of rhombomere 5 affects cochlear duct growth, while rhombomere 6 removal affects cochlear growth and morphogenesis. Rotating rhombomeres 5 and 6 along the anteroposterior axis also impacts cochlear duct morphogenesis but has little effect on the vestibular components. Our studies indicate that discrete tissues, acting at a distance, control the morphogenesis of distinct elements of the inner ear. These results provide a basis for identifying factors that are essential to vestibular and auditory development in vertebrates.     

Mouse models to study inner ear development and hereditary hearing loss

Hereditary sensorineural hearing loss, derived from inner ear defects, is the most common hereditary disability with a prevalence of 1 in 1,000 children, although it can be present in up to 15% of births in isolated communities. The mouse serves as an ideal animal model to identify new deafness-related genes and to study their roles in vivo. This review describes mouse models for genes that have been linked with hearing impairment (HI) in humans. Mutations in several groups of genes have been linked with HI in both mice and humans. Mutant mice have been instrumental in elucidating the function and mechanisms of the inner ear. For example, the roles of collagens and tectorins in the tectorial membrane, as well as the necessity of intact links between the hair cell projections, stereocilia and kinocilia, have been discovered in mice. Accurate endolymph composition and the proteins which participate in its production were found to be crucial for inner ear function, as well as several motor proteins such as prestin and myosins. Two systematic projects, KOMP and EUCOMM, which are currently being carried out to create knock-out and conditional mutants for every gene in the mouse genome, promise that many additional deafness-related genes will be identified in the next years, providing models for all forms of human deafness.     

An enhancer-based gene-therapy strategy for spatiotemporal control of cargoes during tissue repair

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Axon guidance in the inner ear

Statoacoustic ganglion (SAG) neurons send their peripheral processes to navigate into the inner ear sensory organs where they will ultimately become post-synaptic to mature hair cells. During early ear development, neuroblasts delaminate from a restricted region of the ventral otocyst and migrate to form the SAG. The pathfinding mechanisms employed by the processes of SAG neurons as they search for their targets in the periphery are the topic of this review. Multiple lines of evidence exist to support the hypothesis that a combination of cues are working to guide otic axons to their target sensory organs. Some pioneer neurites may retrace their neuronal migratory pathway back to the periphery, yet additional guidance mechanisms likely complement this process. The presence of chemoattractants in the ear is supported by in vitro data showing that the otic epithelium exerts both trophic and tropic effects on the statoacoustic ganglion. The innervation of ectopic hair cells, generated after gene misexpression experiments, is further evidence for chemoattractant involvement in the pathfinding of SAG axons. While the source(s) of chemoattractants in the ear remains unknown, candidate molecules, including neurotrophins, appear to attract otic axons during specific time points in their development. Data also suggest that classical axon repellents such as Semaphorins, Eph/ephrins and Slit/Robos may be involved in the pathfinding of otic axons. Morphogens have recently been implicated in guiding axonal trajectories in many other systems and therefore a role for these molecules in otic axon guidance must also be explored.     

Cell lines in inner ear research

Cell lines have provided important experimental tools that have enhanced our understanding of neural and sensory function. They are particularly valuable in inner ear research because the auditory and vestibular systems are small, complex, and encased in several layers of bone. Organotypic cultures provide an invaluable experimental resource but require repeated microdissection and culture, and remain complex in terms of cell types and states of differentiation. A number of laboratories have established cell lines that offer a range of potential applications to hearing research. This review describes the advances that have already been made with these lines and the potential applications that they offer in the future. The majority of the cell lines are immortalized with a conditionally expressed, temperature sensitive variant of the SV40 tumor antigen. We discuss the value of these cells in developmental studies.     

Molecular mechanisms of inner ear development

The inner ear is a structurally complex vertebrate organ built to encode sound, motion, and orientation in space. Given its complexity, it is not surprising that inner ear dysfunction is a relatively common consequence of human genetic mutation. Studies in model organisms suggest that many genes currently known to be associated with human hearing impairment are active during embryogenesis. Hence, the study of inner ear development provides a rich context for understanding the functions of genes implicated in hearing loss. This chapter focuses on molecular mechanisms of inner ear development derived from studies of model organisms.     

Effects of noise on inner ear

The effects of noise on health depend both on individual factors and characteristics of sound exposure. In case of acoustic trauma, reversible or irreversible lesions of inner ear components are possible. Most often there is immediately an acute tinnitus and hearing loss. Audiometric tests demonstrate hearing loss on the high frequency, generally focused on 4 kHz. Immediate treatment is recommended even there is no currently indicator of the ability to restore hearing. New perspectives on treatment are directed to local treatment and/or using new procedure as antioxidative treatment. Occupational and leisure are the two conditions in which chronic exposure to noise is found. Detection and prevention of noise-induced hearing loss is easier in case of industrial workers than in case of noise exposition for musicians and other sounds and stage technicians or concert managers, and of course non-professional with exposure to amplified music.     

Molecular screens for inner ear genes

Identification of the genes that encode proteins that are important for proper function of specific inner ear cell types is central to our understanding of the molecular basis of hearing and balance. Whereas the combination of electrophysiology and biophysics has resulted in an exquisite understanding of inner ear function, little is known about the proteins that confer these properties at the cellular level. Furthermore, the genes that control inner ear development, susceptibility to wear and tear, regeneration from damage, and age-related degeneration, are largely unknown. This review discusses tools that have been developed during the past few years to address this imbalance between a thorough physiologic characterization of inner ear function and a detailed understanding at a molecular level of the proteins involved in these functions. Creation of inner ear cDNA libraries has laid the foundation for the discovery of genes that are specifically expressed by cell types of the inner ear and that encode proteins that are important for molecular processes in these cells. In conjunction with expressed sequence tag database analysis, cDNA subtraction, and DNA arrays, functionally important genes, whose specific expression patterns are usually verified by gene expression analysis, can be identified. Discussion of these techniques takes into account the specific characteristics of the inner ear in relation to its study using molecular biological approaches.