Leonor Santos-Ruiz

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.

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.

Osteogenic molecules for clinical applications: improving the BMP-collagen system.

Among the osteogenic growth factors used for bone tissue engineering, bone morphogenetic proteins (BMPs) are the most extensively studied for use in orthopaedic surgery. BMP-2 and BMP-7 have been widely investigated for developing therapeutic strategies and are the only two approved for use in several clinical applications. Due to the chemical and biological characteristics of these molecules, their authorised uses are always in combination with a carrier based on collagen type I. Although the use of these growth factors is considered safe in the short term, the very high doses needed to obtain significant osteoinduction make these treatments expensive and their long-term safety uncertain, since they are highly pleiotropic and have the capacity to induce ectopic ossification in the surrounding tissues. Therefore it is necessary to improve the currently used BMP-collagen system in terms of efficiency, biosecurity and costs. There are several strategies to increase the clinical effectiveness of these treatments. In this review we summarize the most promising results and our related work focused on this field through two different approaches: i) the development of recombinant BMPs with additional features, and ii) complementing these systems with other growth factors or molecules to enhance or accelerate osteogenesis.

Basic fibroblast growth factor enhances the osteogenic differentiation induced by bone morphogenetic protein-6 in vitro and in vivo.

Some members of the bone morphogenetic protein subfamily (BMP-2 and -7) are currently used in orthopedic surgery for several applications. Although their use is considered safe at short term, the high doses of growth factors needed make these treatments expensive and their safety uncertain at long term. BMP-6 has been much less studied than BMP-2 and -7, but some authors suggest that this BMP might have a stronger osteogenic activity than the previously mentioned. Having in mind that angiogenesis plays a well-known role during bone formation, the aim of this work was to study the effect of combining BMP-6 with bFGF on both the growth and differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs), as well as on in vivo osteogenesis. We demonstrate that a low dose of bFGF enhances the osteogenic differentiation of MSCs induced by BMP-6 in vitro. Furthermore, we also demonstrate that bone formation in vivo induced by BMP-6 can be accelerated and enhanced by adding a low dose of bFGF, what might suggest a synergic effect between these growth factors on in vivo osteogenesis.

Differential regulation of fibroblast growth factor receptors in the regenerating amphibian spinal cord in vivo

Unlike mammals, adult urodele amphibians can regenerate their spinal cord and associated ganglia, but the molecular mechanisms controlling regeneration are not fully understood. We have recently shown that expression of FGF2, a member of the fibroblast growth factor family, is induced in the progenitor cells of the regenerating spinal cord and appears to play a role in their proliferation and possibly in their differentiation. In order to investigate which receptor(s) may mediate FGF2 signaling and their role in regeneration, we have studied expression of the four fibroblast growth factor receptors, FGFR1, FGFR2, FGFR3 and FGFR4, and of the spliced variants, sFGFR and KGFR, in the regenerating spinal cord of the adult urodele, Pleurodeles waltl, following tail amputation. We show that all FGFRs are expressed in normal and regenerating spinal cord, with the exception of the spliced variants that are expressed only in non-neural tissues of the tail. FGFR1 and 4 show the more interesting spatio-temporal patterns of expression. They are not detectable in the ependymal cells of normal cords, from which neural progenitors for regeneration are believed to originate, though they are expressed in some mature neurons. During regeneration, significant up-regulation of FGFR1 precedes that of FGFR4 in the ependymal tube from which the new cord will form. FGFR4 is highly expressed in these cells at later stages of regeneration, when neuronal differentiation is becoming apparent, and like FGFR1 is also expressed in some newborn neurons. In addition to the known form of FGFR1, the antibody against this receptor reacts also with a non-phosphorylated protein that appears to be present only during regeneration, and might represent a yet undescribed variant of the receptor. Altogether this study shows that fibroblast growth factor signaling is finely modulated during tail and spinal cord regeneration, and points to FGFR1 and FGFR4 as key players in this process, suggesting that FGFR1 is primarily associated with proliferation of progenitor cells and FGFR4 with early stages of neuronal differentiation.

A single-point mutation in FGFR2 affects cell cycle and Tgfβ signalling in osteoblasts

Fgf and Tgfbeta are key regulators of bone development. It is not known, however, whether there is a relationship between defective Fgf signalling, resulting in a premature cranial suture fusion, and Tgfbeta signalling. We used mouse calvaria osteoblasts carrying a mutation (hFGFR2-C278F) associated with Crouzon and Pfeiffer syndromes to investigate effects of this mutation on cell growth and possible mechanisms underlying it. Mutated osteoblasts displayed reduced S-phase, increased apoptosis and increased differentiation. As Tgfbeta signalling appeared to be required in an autocrine/paracrine manner for osteoblast proliferation, we tested the hypothesis that reduced growth might be due, at least in part, to an altered balance between FGF and Tgfbeta signalling. Tgfbeta expression was indeed decreased in mutated osteoblasts, as compared to osteoblasts carrying the wild type hFGFR2. Treatment with Tgfbeta, however, neither increased proliferation in mutated osteoblasts, unlike in controls, nor rescued proliferation in control osteoblasts treated with an Erk1/2 inhibitor. Significantly, Erk2, that is important for proliferation, was reduced relatively to Erk1 in mutated cells. Altogether this study suggests that the hFGFR2-C278F mutation affects the osteoblast ability to respond to Tgfbeta stimulation via the Erk pathway and that the overall effect of the mutation is a loss of function.

Osteoprogenitor cell adhesiveness to a titanium mesh. A clinically relevant hypothesis for revision surgery in hip replacement

Bone defects are a major problem in primary and, even worse, revision surgery of the hip. Impacted allograft contained by a metallic mesh has been used for bone reconstruction. However, since allograft has been claimed to reabsorb, and to provoke immunogenic responses with potential viral infection and loosening because of allograft and mesh interface, new bone substitutes are being searched for. In this study we have cultured committed osteoprogenitor cells within a titanium mesh. The cells not only multiplied very easily, but also adhered to the mesh surface. We believe this phenomenon may have great clinical relevance for cell therapy as an alternative to allograft for the treatment of bone defects in acetabular or femoral reconstruction in revision surgery of the hip.

A collagen-targeted biomimetic RGD peptide to promote osteogenesis.

Osteogenesis is a complex, multifactorial process in which many different signals interact. The bone morphogenetic proteins (BMPs) are the most potent inducers of osteoblastic differentiation, although very high doses of BMPs in combination with collagen type I formulations have to be used for clinical applications. Although integrin-binding arginine-glycine-aspartic acid (RGD) biomimetic peptides have shown some promising abilities to promote the attachment of cells to biomaterials and to direct their differentiation, the linking of these peptides to collagen sponges usually implies chemical manipulation steps. In this study, we describe the design and characterization of a synthetic collagen-targeted RGD biomimetic (CBD-RGD) peptide formed from a collagen-binding domain derived from the von Willebrand factor and the integrin-binding RGD sequence. This peptide was demonstrated to bind to absorbable collagen type I sponges (ACSs) without performing any chemical linking, and to induce the differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells. Furthermore, in vivo experiments showed that ACSs functionalized with CBD-RGD and loaded with a subfunctional dose of BMP-2-formed ectopic bone in rats, while nonfunctionalized sponges loaded with the same amount of BMP-2 did not. These results indicate that the combination of this biomimetic peptide with the currently used collagen+BMP system might be a promising approach to improve osteogenesis and to reduce the doses of BMPs needed in clinical orthopedics.