José Becerra

Old questions, new tools, and some answers to the mystery of fin regeneration†

Pluridisciplinary approaches led to the notion that fin regeneration is an intricate phenomenon involving epithelial–mesenchymal and reciprocal exchanges throughout the process as well as interactions between ray and interray tissue. The establishment of a blastema after fin amputation is the first event leading to the reconstruction of the missing part of the fin. Here, we review our knowledge on the origin of the blastema, its formation and growth, and of the mechanisms that control differentiation and patterning of the regenerate. Our current understanding results from studies of fin regeneration performed in various teleost fish over the past century. We also report the recent breakthroughs that have been made in the past decade with the arrival of a new model, the zebrafish, Danio rerio, which now offers the possibility to combine cytologic, molecular, and genetic analyses and open new perspectives in this field. Developmental Dynamics 226:190–201, 2003.

Skeletal deformities in larval, juvenile and adult stages of cultured gilthead sea bream (Sparus aurata L.)

Abstract Gilthead sea bream (Sparus aurata L.) is currently farmed in a number of European countries. In culture exploitations, spinal malformations are frequently seen in adult specimens. We studied skeletal deformities in larval, juvenile and adult stages of fish from a Spanish experimental culturing centre. About 27% of sea bream larvae at hatching showed different types of axial deformations that were related to notochord alterations during embryogenesis. About 22% died soon after hatching, but 5% survived and reached juvenile and adult stages. These fish were mostly lordotic. Juvenile lordotic fish displayed uninflated swimbladders but all lordotic adults possessed an inflated functional swimbladder. Lordosis is characterised by V-shaped dorsoventral curvature of the body axis including the vertebral column and the spinal cord. Spinal curvature occurred more frequently between vertebrae 10 and 16. The congenital or postnatal origin of lordosis is discussed.

Structure of the tail fin in teleosts

SummaryA morphologic study of the structure of the tail fin in eight species of teleosts was performed by aid of the Picrosirius-polarization method, which is a specific histochemical method for the detection of collagen in tissue sections. This structure is composed mainly of skeletal elements, the fin rays, covered by skin. Fin rays are bound to each other and to the surrounding tissues by a series of collagenous ligaments forming a complex, highly pliable and resistant structure. Although the general structural pattern of tail fins was consistent in all species studied, the comparative aspects reported in this paper show that variations in the form and size of their components are responsible for the morphologic diversities which are closely related to specific functional adaptations. Morphometric data on the number and size of actinotrichia in normal adult specimens are presented.

The effect of an rhBMP-2 absorbable collagen sponge-targeted system on bone formation in vivo

Reparation of bone defects remains a major clinical and economic concern, with more than 3 million bone grafts performed annually only in the United States and the EU. The search for alternatives to autologous bone grafting led to the approval by the FDA of an absorbable collagen carrier combined with rhBMP-2 for the treatment of certain bone diseases and fractures. The present work is focused on the production of a collagen-targeted rhBMP-2 based system to improve bone formation. We produced a modified rhBMP-2 with only an additional collagen-binding decapeptide derived from the von Willebrand factor and tested its affinity to collagen and its ability to induce ectopic bone formation in vivo when implanted in combination with absorbable collagen sponges or hydroxyapatite. The results showed not only that the rhBMP2-CBD had an increased affinity to collagen, but also that this binding was very stable during a prolonged period of time. In vivo experiments demonstrated that this rhBMP2-CBD maintained its osteoinductive activity, being capable of inducing new bone formation even at lower concentrations than native rhBMP-2. These results indicate that the combination of the fusion protein with absorbable collagen may be a suitable and safer alternative to rhBMP-2 for bone repair purposes.

Cell proliferation during blastema formation in the regenerating teleost fin.

Epimorphic regeneration in teleost fins occurs through the establishment of a balanced growth state in which a blastema gives rise to all the mesenchymal cells, whereas definite areas of the epidermis proliferate leading to its extension, thus, allowing the enlargement of the whole structure. This type of regeneration involves specific mechanisms that temporally and spatially regulate cell proliferation. To understand how the blastema is formed and how this growth situation is set up, we investigated cell proliferation patterns in the regenerating fin of the goldfish Carassius auratus from the time of amputation to that of blastema formation by using proliferating cell nuclear antigen immunostaining and bromodeoxyuridine labeling. Wound closure and apical epidermal cap formation took place by epidermal migration and re‐arrangement, without the contribution of cell proliferation. As soon as the apical cap had formed, the epidermis started to proliferate at its lateral surfaces, in which all layers maintained cycling for the duration of the studied process. The distal epidermal cap, on the contrary, presented very few cycling cells, and its cytoarchitecture was indicative of continuous remodeling due to ray growth. The basal layer of this epidermal cap showed a typical morphology and remained nonproliferative whilst in contact with the proliferating blastema. Proliferation in the mesenchymal compartment of the ray started far from the amputation plane. Subsequently, cycling cells approached that location, until they formed the blastema in contact with the apical epidermal cap. Differences observed between the epidermis and mesenchyma, regarding activation of the cell cycle and the establishment of proliferative patterns, suggest that differential mechanisms regulate cell proliferation in each of these compartments during the initial stages of regeneration.

The Stem Cell Niche Should be a Key Issue for Cell Therapy in Regenerative Medicine

Recent advances in stem cell research have highlighted the role played by such cells and their environment (the stem cell niche) in tissue renewal and homeostasis. The control and regulation of stem cells and their niche are remaining challenges for cell therapy and regenerative medicine on several tissues and organs. These advances are important for both, the basic knowledge of stem cell regulation, and their practical translational applications into clinical medicine. This article is primarily concerned with the mesenchymal stem cells (MSCs) and it reviews the current aspects of their own niche. We discuss on the need for a deeper understanding of the identity of this cell type and its microenvironment in order to improve the effectiveness of any cell therapy for regenerative medicine. Ex vivo reproduction of the conditions of the natural stem cell niche, when necessary, would provide success to tissue engineering. The first challenge of regenerative medicine is to find cells able to replace and/or repair the lost function of tissues and organs by disease or aging and the trophic and immunomodulatory effects recently found for MSCs open up for new opportunities. If MSCs are pericytes, as it has been proposed, perhaps it may explain the ubiquity of these cells and their possible role in miscellaneous repairs throughout the body opening for new chances for extensive tissue repair.

Actinotrichia collagens and their role in fin formation.

The skeleton of zebrafish fins consists of lepidotrichia and actinotrichia. Actinotrichia are fibrils located at the tip of each lepidotrichia and play a morphogenetic role in fin formation. Actinotrichia are formed by collagens associated with non-collagen components. The non-collagen components of actinotrichia (actinodins) have been shown to play a critical role in fin to limb transition. The present study has focused on the collagens that form actinotrichia and their role in fin formation. We have found actinotrichia are formed by Collagen I plus a novel form of Collagen II, encoded by the col2a1b gene. This second copy of the collagen II gene is only found in fishes and is the only Collagen type II expressed in fins. Both col1a1a and col2a1b were found in actinotrichia forming cells. Significantly, they also expressed the lysyl hydroxylase 1 (lh1) gene, which encodes an enzyme involved in the post-translational processing of collagens. Morpholino knockdown in zebrafish embryos demonstrated that the two collagens and lh1 are essential for actinotrichia and fin fold morphogenesis. The col1a1 dominant mutant chihuahua showed aberrant phenotypes in both actinotrichia and lepidotrichia during fin development and regeneration. These pieces of evidences support that actinotrichia are composed of Collagens I and II, which are post-translationally processed by Lh1, and that the correct expression and assembling of these collagens is essential for fin formation. The unique collagen composition of actinotrichia may play a role in fin skeleton morphogenesis.

Engineering, Expression, and Renaturation of a Collagen-Targeted Human bFGF Fusion Protein

Abstract Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen-binding domain, and (iii) cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains a collagen-binding domain), located in inclusion bodies, were solubilized with 6M guanidine-HCI and renatured using a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factors was demonstrated by their ability to stimulate proliferation of human vein endothelial cells (HVEC), monitored by [3H]-thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. Binding of the renatured hbFGF-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of [3H]-collagen to the rhbFGF-F2 protein immobilized on a Ni-NTA column. The rhbFGF-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen-binding domain effectively targets the recombinant growth factor to type I collagen. The clinical effect of rhbFGF-F2 on wound healing is also studied in streptozotocin-induced diabetic rats and evaluated by histological examination comparing with rhbFGF-F1 and commercial bFGF effects. The highly beneficial effects of rhbFGF-F2 on wound healing is suggested to be due to its extremely potent angiogenesis and granulation tissue formation activities, leading to a rapid reepithelialization of the wound. Topical application of rhbFGF-F2 mixed with type I collagen is a more effective method in accelerating closure of full-thickness excisional skin-wound in diabetic rats when compared with the fusion protein alone or commercial hbFGF at the same doses. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins as well as to develop new strategies for specific biomedical applications.

Dual luciferase labelling for non-invasive bioluminescence imaging of mesenchymal stromal cell chondrogenic differentiation in demineralized bone matrix scaffolds

Non-invasive bioluminescence imaging (BLI) to monitor changes in gene expression of cells implanted in live animals should facilitate the development of biomaterial scaffolds for tissue regeneration. We show that, in vitro, induction of chondrogenic differentiation in mouse bone marrow stromal cell line (CL1) and human adipose tissue derived mesenchymal stromal cells (hAMSCs), permanently transduced with a procollagen II (COL2A1) promoter driving a firefly luciferase gene reporter (PLuc) (COL2A1p.PLuc), induces PLuc expression in correlation with increases in COL2A1 and Sox9 mRNA expression and acquisition of chondrocytic phenotype. To be able to simultaneously monitor in vivo cell differentiation and proliferation, COL2A1p.PLuc labelled cells were also genetically labelled with a renilla luciferase (RLuc) gene driven by a constitutively active cytomegalovirus promoter, and then seeded in demineralized bone matrix (DBM) subcutaneously implanted in SCID mice. Non-invasive BLI monitoring of the implanted mice showed that the PLuc/RLuc ratio reports on gene expression changes indicative of cell differentiation. Large (CL1) and moderated (hAMSCs) changes in the PLuc/RLuc ratio over a 6 week period, revealed different patterns of in vivo chondrogenic differentiation for the CL1 cell line and primary MSCs, in agreement with in vitro published data and our results from histological analysis of DBM sections. This double bioluminescence labelling strategy together with BLI imaging to analyze behaviour of cells implanted in live animals should facilitate the development of progenitor cell/scaffold combinations for tissue repair.

Zebrafish fins as a model system for skeletal human studies.

Recent studies on the morphogenesis of the fins of Danio rerio (zebrafish) during development and regeneration suggest that a number of inductive signals involved in the process are similar to some of those that affect bone and cartilage differentiation in mammals and humans. Akimenko et al. (2002) has shown that bone morphogenetic protein-2b (BMP2b) is involved in the induction of dermal bone differentiation during fin regeneration. Many other groups have also shown that molecules from the transforming growth factor-beta superfamily (TGFβ), including BMP2, are effective in promoting chondrogenesis and osteogenesis in vivo in higher vertebrates, including humans. In the present study, we review the state of the art of this topic by a comparative analysis of skeletal tissue development, regeneration and renewal processes in tetrapods, and fin regeneration in fishes. A general conclusion of this study states that lepidotrichia is a special skeletal tissue different to cartilage, bone, enamel, or dentine in fishes, according to its extracellular matrix (ECM) composition. However, the empirical analysis of inducing signals of skeletal tissues in fishes and tetrapods suggests that lepidotrichia is different to any responding features with main skeletal tissues. A number of new inductive molecules are arising from fin development and regeneration studies that might establish an empirical basis for further molecular approaches to mammal skeletal tissues differentiation. Despite the tissue dissimilarity, this empirical evidence might finally lead to clinical applications to skeletal disorders in humans.