Generation and Evaluation of Novel Biomaterials Based on Decellularized Sturgeon Cartilage for Use in Tissue Engineering.

Because cartilage has limited regenerative capability, a fully efficient advanced therapy medicinal product is needed to treat severe cartilage damage. We evaluated a novel biomaterial obtained by decellularizing sturgeon chondral endoskeleton tissue for use in cartilage tissue engineering. In silico analysis suggested high homology between human and sturgeon collagen proteins, and ultra-performance liquid chromatography confirmed that both types of cartilage consisted mainly of the same amino acids. Decellularized sturgeon cartilage was recellularized with human chondrocytes and four types of human mesenchymal stem cells (MSC) and their suitability for generating a cartilage substitute was assessed ex vivo and in vivo. The results supported the biocompatibility of the novel scaffold, as well as its ability to sustain cell adhesion, proliferation and differentiation. In vivo assays showed that the MSC cells in grafted cartilage disks were biosynthetically active and able to remodel the extracellular matrix of cartilage substitutes, with the production of type II collagen and other relevant components, especially when adipose tissue MSC were used. In addition, these cartilage substitutes triggered a pro-regenerative reaction mediated by CD206-positive M2 macrophages. These preliminary results warrant further research to characterize in greater detail the potential clinical translation of these novel cartilage substitutes.

The metabolic effects of prolonged starvation and refeeding in sturgeon and rainbow trout.

The present study examines the particular metabolic strategies of the sturgeon Acipenser naccarii in facing a period of prolonged starvation (72 days) and subsequent refeeding (60 days) compared to the trout Oncorhynchus mykiss response under similar conditions. Plasma metabolites, endogenous reserves, and the activity of intermediate enzymes in liver and white muscle were evaluated. This study shows the mobilization of tissue reserves during a starvation period in both species with an associated enzymatic response. The sturgeon displayed an early increase in hepatic glycolysis during starvation. The trout preferentially used lactate for gluconeogenesis in liver and white muscle. The sturgeon had higher lipid-degradation capacity and greater synthesis of hepatic ketone bodies than the trout, although this latter species also showed strong synthesis of ketone bodies during starvation. During refeeding, the metabolic activity present before starvation was recovered in both fish, with a reestablishment of tissue reserves, plasmatic parameters (glucemia and cholesterol), and enzymatic activities in the liver and muscle. A compensatory effect in enzymes regarding lipids, ketone bodies, and oxidative metabolism was displayed in the liver of both species. There are metabolic differences between sturgeon and trout that support the contention that the sturgeon has common characteristics with elasmobranchs and teleosts.

The development of the epicardium in the sturgeon Acipenser naccarii.

This article reports on the development of the epicardium in alevins of the sturgeon Acipenser naccarii, aged 4-25 days post-hatching (dph). Epicardial development starts at 4 dph with formation of the proepicardium (PE) that arises as a bilateral structure at the boundary between the sinus venosus and the duct of Cuvier. The PE later becomes a midline organ arising from the wall of the sinus venosus and ending at the junction between the liver, the sinus venosus and the transverse septum. This relative displacement appears related to venous reorganization at the caudal pole of the heart. The mode and time of epicardium formation is different in the various heart chambers. The conus epicardium develops through migration of a cohesive epithelium from the PE villi, and is completed through bleb-like aggregates detached from the PE. The ventricular epicardium develops a little later, and mostly through bleb-like aggregates. The bulbus epicardium appears to derive from the mesothelium located at the junction between the outflow tract and the pericardial cavity. Strikingly, formation of the epicardium of the atrium and the sinus venosus is a very late event occurring after the third month of development. Associated to the PE, a sino-ventricular ligament develops as a permanent connection. This ligament contains venous vessels that communicate the subepicardial coronary plexus and the sinus venosus, and carries part of the heart innervation. The development of the sturgeon epicardium shares many features with that of other vertebrate groups. This speaks in favour of conservative mechanisms across the evolutionary scale.

Effect of starvation and refeeding on digestive enzyme activities in sturgeon (Acipenser naccarii) and trout (Oncorhynchus mykiss).

The digestive enzyme activities were determined in Adriatic sturgeon and rainbow trout during starvation and refeeding period. Overall, the digestive enzyme activities are affected in the same sense in both species. The protease and lipase activities were decreased later than amylase activity. Even after 1 month of starvation, both species would be prepared to digest protein and lipids in an effective way. After 72 days of starvation, the digestive machinery of the sturgeon and of the trout shows an altered capacity to digest macronutrients. The capacity to digest proteins and lipids, after 60 days of refeeding, begins to become re-established in sturgeon and trout. In contrast, in this period, the capacity to digest carbohydrates remains depressed in both species.

Structural characteristics and development of ampullary organs in Acipenser naccarii.

Ampullary organs of Acipenser naccarii sturgeons were examined by optical and electronic microscopy (transmission electron microscopy and scanning electron microscopy) from hatching until 1 month later when the juvenile phase is completely established. It was observed that, when A. naccarii begins to feed actively, the ultrastructural characteristics of ampullary organs already correspond to those of adult animals. These organs may, therefore, be functional and, together with taste buds, facilitate food search after exhaustion of yolk sac food reserves. Mature ampullary organs of A. naccarii are formed by an ampulla that communicates with the exterior by means of a short channel. These ampullae correspond to the sensory portion of these receptors and are formed by two cell types: receptor cells and support cells. Receptor cells present a kinocilium on their free surface and establish ribbon synapses with axon nerve endings that arise from the underlying conjunctive tissue. Support cells enclose receptor cells, bear stereocilia and occasional cilia, and are of a secretory nature. The mucus associated with ampullary organs mainly comprises neutral mucopolysaccharides, whereas mucopolysaccharides are usually acid in other fish groups.

Dorsoventral transposition of the heart chambers in sturgeon Acipenser naccarii alevins.

This is the first description of a dorsoventral transposition of the heart chambers in sturgeons Acipenser naccarii. The affected individuals were 2 farmed alevins aged 9 and 10 d posthatching, respectively. One was examined by light microscopy and the other by scanning electron microscopy. In both cases, the atrium and sinus venosus occupied a left ventrolateral position, the ventricle, conus arteriosus and bulbus arteriosus were located dorsally, and the transverse septum was incomplete. The anomalous heart examined by light microscopy did not differ histologically from normal hearts of similar developmental stages. The abnormal dorsoventral arrangement of the heart chambers was presumably due to a distortion of the morphogenetic movements that bring the ventricle to the ventral and the atrium to the dorsal position. The present findings, together with genetic data reported in the literature, suggest that the defective cardiac phenotype detected in the present specimens might result from a mutation affecting the sturgeon ortholog of the zebrafish overlooped (olp) gene.

Adaptive branchial mechanisms in the sturgeon Acipenser naccarii during acclimation to saltwater.

Variations of Na(+)/K(+)-ATPase activity and fatty-acid composition in the gills of the sturgeon Acipenser naccarii subjected to progressive acclimation to full seawater (35 ppt) were determined in relation to the hypo-osmoregulatory capacity of this species in the hyperosmotic medium. Blood samples were taken and gills arches were removed at intermediate salinity levels between 0 and 35 ppt and after 20 days at constant salinity (35 ppt). Plasma osmolality and Na(+)/K(+)-ATPase activity increased significantly with growing environmental salinity. Total saturated fatty acids (SFAs) decreased, while total polyunsaturated fatty acids (PUFAs) increased significantly with increasing salinity due mainly to changes in n-3 PUFAs (20:5n-3 and 22:6n-3). The n-3/n-6 ratio increased significantly during the acclimation process. The results show a direct relationship between salinity, increased gill Na(+)/K(+)-ATPase activity and ultrastructural changes of the gill chloride cells. Changes in the fatty-acid composition in gills of A. naccarii during progressive acclimation to full seawater suggest that variations of gill fatty acids may also have a role in osmoregulatory mechanisms.

The development of the sturgeon heart.

This paper presents a sequential analysis of the development of the sturgeon (Acipenser naccarii) heart from the end of gastrulation to the early juvenile stages. At late neurulation, the heart appears as a straight, short tube located over the endoderm that forms the wall of the yolk sac, in front of the developing head. The heart axis is aligned with the axis of the developing head. Subsequently, the heart elongates and adopts a C-shape, and its axis becomes perpendicular to that of the head. Around the time of hatching, the heart loses the loop and appears as a mostly straight tube with the chambers arranged in a craniocaudal sequence: outflow tract, ventricle, atrium, and a small sinus venosus. During the first 4 days post-hatching (dph), the heart starts looping again, adopts a C-shape, and undergoes a counterclockwise movement that brings the atrium to the left of the outflow tract and the ventricle to a caudal position. Thus, a primary and a secondary cardiac loop occur in the sturgeon. Later, the atria come to occupy a middle position behind the outflow tract, and the sinus venosus shifts from a caudal to a dorsal position. A morphological arrangement similar to that found in adult sturgeons is attained in all specimens at days 7-9 dph. The external changes are accompanied by a series of internal modifications that include trabeculation (3-4 dph), development of endocardial cushions in the atrioventricular canal (4 dph) and in the conus arteriosus (3-4 dph), conus (22-24 dph) and atrioventricular (18-20 dph) valve formation, and development of the epicardium (4 dph) and the coronary vessels (10 dph). The main developmental features of the heart have been registered, and a basic body of information, which should be very useful in future developmental studies, has been established. Similarities and dissimilarities between the development of the sturgeon heart and that of other vertebrates are underscored.

Differentiation of the cardiac outflow tract components in alevins of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes): implications for heart evolution.

Previous work showed that in the adult sturgeon an intrapericardial, nonmyocardial segment is interposed between the conus arteriosus of the heart and the ventral aorta. The present report illustrates the ontogeny of this intermediate segment in Acipenser naccarii. The sample studied consisted of 178 alevins between 1 and 24 days posthatching. They were examined using light and electron microscopy. Our observations indicate that the entire cardiac outflow tract displays a myocardial character during early development. Between the fourth and sixth days posthatching, the distal portion of the cardiac outflow tract undergoes a phenotypical transition, from a myocardial to a smooth muscle-like phenotype. The length of this region with regard to the whole outflow tract increases only moderately during subsequent developmental stages, becoming more and more cellularized. The cells soon organize into a pattern that resembles that of the arterial wall. Elastin appears at this site by the seventh day posthatching. Therefore, two distinct components, proximal and distal, can be recognized from the fourth day posthatching in the cardiac outflow tract of A. naccarii. The proximal component is the conus arteriosus, characterized by its myocardial nature and the presence of endocardial cushions. The distal component transforms into the intrapericardial, nonmyocardial segment mentioned above, which is unequivocally of cardiac origin. We propose to designate this segment the "bulbus arteriosus" because it is morphogenetically equivalent to the bulbus arteriosus of teleosts. The present findings, together with data from the literature, point to the possibility that cells from the cardiac neural crest are involved in the phenotypical transition that takes place at the distal portion of the cardiac outflow tract, resulting in the appearance of the bulbus arteriosus. Moreover, they suggest that the cardiac outflow tract came to be formed by a bulbus arteriosus and a conus arteriosus from an early period of the vertebrate evolutionary story. Finally, we hypothesize that the embryonic truncus of birds and mammals is homologous to the bulbus arteriosus of fish.

Renal corpuscle of the sturgeon kidney: an ultrastructural, chemical dissection, and lectin-binding study.

The sturgeon is an ancient species of fish that thrives in a wide range of ecological environments, from freshwater to seawater. Basic in this process of adaptation is the ability of the kidney to control fluid filtration and urine formation. However, the morphological basis of this process is mostly unknown. The aim of the present study was to use microdissection techniques (scanning electron microscopy (SEM), transmission electron microscopy (TEM), and lectin-binding histochemistry) to examine the structure of the renal corpuscle of the sturgeon Acipenser nacarii in order to reveal morphologic features that could be related to function, phylogeny, and habitat. The renal corpuscles are aligned along the intrarrenal arteries. The urinary pole shows a siphon-like neck segment (NS) in 92% of the nephrons, whose structural characteristics are different from those of other fish. The podocytes have cuboidal cellular bodies, intercellular contacts, and poorly developed cell processes. The podocyte glycocalyx contains N-acetylglucosamine and lacks sialic acid. The structural and lectin-binding patterns are similar to those found in the immature mammalian kidney. The glomerular basement membrane (GBM) is very thick and consists of three layers: a lamina rara externa, a lamina densa, and a thick subendothelial lamina. The latter contains tubular microfibrils, collagen fibers, and long mesangial cell processes. Frequently, the podocyte bodies attach directly to the GBM, and the area occupied by the filtration slits is very small. Furthermore, the GBM shows a glycosylation pattern different from that observed in most vertebrates. Contrary to what would be expected in sturgeons living in freshwater, the A. nacarii renal corpuscle morphology suggests a low glomerular filtration rate.

Cleared extrachromosomal domain (CED): a nuclear domain enriched in nuclear matrix filaments is a common structure in sturgeon podocytes.

The cell nucleus is divided into chromosome territories and the extrachromosomal domain. The latter includes several structural and functional compartments involved in RNA processing and transport. Morphological and cytochemical analyses of the cell nucleus in sturgeon podocytes revealed the existence of a previously uncharacterised nuclear compartment. It appears as a cleared nucleoplasmic area of variable size within the extrachromosomal domain. Conventional light and electron microscopy revealed that this compartment, here referred to as cleared extrachromosomal domain (CED), appears free of chromatin and RNA-containing organelles and is closely surrounded by heterochromatin masses. Cytochemical and immunogold electron microscope studies indicated that CEDs lack DNA, RNA and glycoconjugates. The ultrastructural examination of Lowicryl-embedded sections showed that CEDs are formed by a fibrillar network. In resinless preparations, this network appears as a dense mesh of crosslinked nuclear matrix filaments. The density of nuclear matrix filaments within the CED is remarkably higher than that observed in the rest of the nucleus. Our results indicate that the CED is a single, distinct extrachromosomal domain of the nuclear matrix of sturgeon podocytes. The absence in the CED of detectable DNA and RNA, as well as the lack of chromatin and RNA-containing structures, suggests that transcription and RNA processing do not occur in this novel nuclear compartment. The volume occupied by the CED may preserve the volume-to-cytoplasm ratio in the podocyte and contribute to modulate the spatial organisation and the volume occupied by the chromosomal territories.