Chitosan sponges as tissue engineering scaffolds for bone formation

Rat calvarial osteoblasts were grown in porous chitosan sponges fabricated by freeze drying. The prepared chitosan sponges had a porous structure with a 100-200 microm pore diameter, which allowed cell proliferation. Cell density, alkaline phosphatase activity and calcium deposition were monitored for up to 56 d culture. Cell numbers were 4 x 10(6) (day 1), 11 x 10(6) (day 28) and 12 x 10(6) (day 56) per g sponge. Calcium depositions were 9 (day 1), 40 (day 28) and 48 (day 56) microg per sponge. Histological results corroborated that bone formation within the sponges had occurred. These results show that chitosan sponges can be used as effective scaffolding materials for tissue engineered bone formation in vitro.     

Development of In Vivo Sponge Cultures: Particle Feeding by the Tropical Sponge Pseudosuberites aff. andrewsi

The rate of food particle uptake of the tropical sponge Pseudosuberites aff. andrewsi was studied in relation to particle concentrations and particle size. A range of different concentrations of either the marine microalga Dunaliella tertiolecta (∼5–8 μm) or the marine cyanobacterium Synechococcus sp. (∼1 μm) was supplied to the sponges. D. tertiolecta had a pronounced effect on the filtration activity of the sponges: at concentrations higher than approximately 4 × 10⁵ cells/cm³, the filtration rates dropped dramatically. Such a clear effect was not found for Synechococcus sp. The results further showed that the maximal amount of food (when expressed in organic carbon) that can be taken up per cubic centimeter of sponge volume per unit of time should in principle be sufficient to enable growth (irrespective of the food particle type). At the maximal food particle concentration that did not affect the filtration rates, the uptake of organic carbon is already highly in excess of the amount of organic carbon that the sponges need to cope with their respiratory demand. Based on these findings, a series of growth experiments was carried out in which the sponges were subjected to a constant concentration of different types of food particles (Synechococcus sp. and the microalgae Chlorella sorokiniana and Nannochloropsis sp). Although initial growth was sometimes observed, continuous growth at a constant rate could not be obtained. It is concluded that qualitative aspects of feeding rather than quantitative aspects are the key to successful in vivo sponge culture. Received December 20, 2000; accepted March 26, 2001     

In Vitro CuIture of the Tropical Sponge <Emphasis Type="Italic">Axinella corrugata</Emphasis> (Demospongiae): Effect of Food Cell Concentration on Growth,Clearance Rate,and Biosynthesis of Stevensine

In vitro culture is one possible method for supplying sponge metabolites for pharmaceutical applications, but appropriate feeding regimens that maximize both growth and metabolite biosynthesis are largely unknown. According to the natural concentration (NC) of cells 1 to 50 µm in size that are available to wild Axinella corrugata, we fed explants a multispecific diet of bacteria, microalgae, and yeast at 4 different concentrations: 1NC, 3NC, 5NC, and 5+1NC (the last consisted of 5 NC of bacteria and 1 NC of microalgae and yeast). Explants fed a 3NC diet had the best culture response, growing on average from 8.5 g to 10.3 g in 8 weeks, and showing a 110% increase in concentration (milligrams per gram of dry weight) of the antitumor compound stevensine. Stevensine production in 3NC explants, representing the total milligrams of metabolite per explant, increased by 157% over the study. Explants fed at 1NC had relatively stable weights, indicating that the diet met metabolic costs only. Explants fed at the two highest concentrations lost weight after 4 weeks, possibly because long-term high cell concentration blocked their aquiferous system, reducing their ability to feed efficiently. Stevensine production in explants fed the 1NC, 5NC, or 5+1NC diets were similar, and varied little from the initial amount. A separate experiment showed that the clearance rate for A. corrugata is similar between the examined food types and cell concentrations over 5 hours, averaging 766 ml h^–1 g DW^–1.Overall, this study demonstrates that relatively small changes in food abundance can greatly affect both sponge growth and metabolite biosynthesis. The good growth and increased production of the target metabolite stevensine for A. corrugata explants fed a 3NC diet suggests that in vitro culture is a viable method of supplying some sponge metabolites.     

Reproductive cycle of the coral-excavating sponge Thoosa mismalolli (Clionaidae) from Mexican Pacific coral reefs

Abstract. Individuals of the recently described demosponge Thoosa mismalolli are common on Mexican Pacific coral reefs, excavating burrows in living corals and in other calcareous substrata. To better understand the propagative abilities of this sponge, we conducted a histological study over an 18-month period (May 2007–November 2008) to identify sexual and asexual reproductive structures. Members of the species are viviparous and hermaphroditic, with various developmental stages of oocytes, spermatic cysts, and embryos co-occurring in the mesohyl for most of the year. This nearly continuous reproductive activity intensified during the warm season. Fertilization was internal, and embryos developed inside the parental sponge to produce an unciliated hoplitomella larva, characterized by a peculiar siliceous skeleton. In addition to the sexually generated larvae, adults of T. mismalolli formed gemmules for asexual reproduction. Gemmules occurred within the mesohyl during all months of the year, but were most abundant in the coldest months. This combination of sexual and asexual processes enables individuals of T. mismalolli to reproduce almost continuously. This strategy may facilitate both long-term persistence within reefs and effective dispersal between distant reefs.     

Metagenomic approaches to exploit the biotechnological potential of the microbial consortia of marine sponges

Natural products isolated from sponges are an important source of new biologically active compounds. However, the development of these compounds into drugs has been held back by the difficulties in achieving a sustainable supply of these often-complex molecules for pre-clinical and clinical development. Increasing evidence implicates microbial symbionts as the source of many of these biologically active compounds, but the vast majority of the sponge microbial community remain uncultured. Metagenomics offers a biotechnological solution to this supply problem. Metagenomes of sponge microbial communities have been shown to contain genes and gene clusters typical for the biosynthesis of biologically active natural products. Heterologous expression approaches have also led to the isolation of secondary metabolism gene clusters from uncultured microbial symbionts of marine invertebrates and from soil metagenomic libraries. Combining a metagenomic approach with heterologous expression holds much promise for the sustainable exploitation of the chemical diversity present in the sponge microbial community.     

Metamorphosis of cinctoblastula larvae (Homoscleromorpha, porifera)

The metamorphosis of the cinctoblastula of Homoscleromorpha is studied in five species belonging to three genera. The different steps of metamorphosis are similar in all species. The metamorphosis occurs by the invagination and involution of either the anterior epithelium or the posterior epithelium of the larva. During metamorphosis, morphogenetic polymorphism was observed, which has an individual character and does not depend on either external or species specific factors. In the rhagon, the development of the aquiferous system occurs only by epithelial morphogenesis and subsequent differentiation of cells. Mesohylar cells derive from flagellated cells after ingression. The formation of pinacoderm and choanoderm occurs by the differentiation of the larval flagellated epithelium. This is possibly due to the conservation of cell junctions in the external surface of the larval flagellated cells and of the basement membrane in their internal surface. The main difference in homoscleromorph metamorphosis compared with Demospongiae is the persistence of the flagellated epithelium throughout this process and even in the adult since exo- and endopinacoderm remain flagellated. The antero-posterior axis of the larva corresponds to the baso-apical axis of the adult in Homoscleromorpha.     

Diversity and functional analysis of luxS genes in Vibrios from marine sponges Mycale laxissima and Ircinia strobilina

Sponges harbor highly diverse and dense microbial communities, providing an environment in which bacterial signaling may be important. Quorum sensing (QS) is a cell density-dependent signaling process that bacteria employ to coordinate and regulate their gene expression. Previous studies have found that bacteria isolated from sponges are able to produce acyl-homoserine lactones (AHLs), an important class of QS molecules found in proteobacteria. Autoinducer-2 (AI-2) is a second class of QS molecule, and is considered to be an interspecies signal. However, AI-2 signaling has not been reported in sponge bacterial symbionts. In this study, degenerate primers were designed based on known Vibrio luxS sequences to amplify the luxS genes encoding AI-2 synthases of several Vibrio isolates from marine sponges Mycale laxissima and Ircinia strobilina. All the vibrios isolated from these two sponges had luxS genes and were able to produce signals with AI-2 activity as detected using a biological reporter. A novel group of luxS sequences was found, thus extending the known diversity of luxS genes. One isolate was chosen for further analysis of its luxS gene by expression of the gene in Escherichia coli DH5α and by characterization of the profile of AI-2 activity. This work provides the first information about luxS genes and AI-2 activity in sponge-associated bacterial communities.     

A bioactive collagen-β tricalcium phosphate scaffold for tissue engineering

Collagen-phosphate composites (COL/β-TCP) are novel materials that have the potential to be used as bone analogues. The aim of our study was to develop a porous bioactive material composed of type I collagen, the main bone protein and tricalcium phosphate, the mineral phase of natural bone, and investigate their in vitro biocompatibility in a human dermal fibroblast culture system. In order to obtain the bioactive materials, type I collagen was isolated from bovine tendon and characterized by physicochemical methods. β-TCP was obtained from calcium carbonate by thermal decomposition at 900 °C temperature. The powder was examined with X-ray diffraction. Two variants of COL/β-TCP scaffolds (P1 and P2) were prepared and examined by scanning electron microscopy. Our results revealed a microporous structure with small white aggregates of β-TCP, non-homogenous scattered in the collagen framework without any preferential orientation. The biocompatibility of the obtained scaffolds was tested by biochemical and histological methods on human fibroblast cultures. Both materials acted as good subtrates for human dermal fibroblast proliferation and migration.     

Diversity and biotechnological potential of the sponge-associated microbial consortia

Sponges are well known to harbor diverse microbes and represent a significant source of bioactive natural compounds derived from the marine environment. Recent studies of the microbial communities of marine sponges have uncovered previously undescribed species and an array of new chemical compounds. In contrast to natural compounds, studies on enzymes with biotechnological potential from microbes associated with sponges are rare although enzymes with novel activities that have potential medical and biotechnological applications have been identified from sponges and microbes associated with sponges. Both bacteria and fungi have been isolated from a wide range of marine sponge, but the diversity and symbiotic relationship of bacteria has been studied to a greater extent than that of fungi isolated from sponges. Molecular methods (e.g., rDNA, DGGE, and FISH) have revealed a great diversity of the unculturable bacteria and archaea. Metagenomic approaches have identified interesting metabolic pathways responsible for the production of natural compounds and may provide a new avenue to explore the microbial diversity and biotechnological potential of marine sponges. In addition, other eukaryotic organisms such as diatoms and unicellular algae from marine sponges are also being described using these molecular techniques. Many natural compounds derived from sponges are suspected to be of bacterial origin, but only a few studies have provided convincing evidence for symbiotic producers in sponges. Microbes in sponges exist in different associations with sponges including the true symbiosis. Fungi derived from marine sponges represent the single most prolific source of diverse bioactive marine fungal compounds found to date. There is a developing interest in determining the true diversity of fungi present in marine sponges and the nature of the association. Molecular methods will allow scientists to more accurately identify fungal species and determine actual diversity of sponge-associated fungi. This is especially important as greater cooperation between bacteriologists, mycologists, natural product chemists, and bioengineers is needed to provide a well-coordinated effort in studying the diversity, ecology, physiology, and association between bacteria, fungi, and other organisms present in marine sponges.     

A modern approach to demineralization of spicules in glass sponges (Porifera: Hexactinellida) for the purpose of extraction and examination of the protein matrix

Glass sponges of the class Hexactinellida are a group of the most ancient multicellular animals, whose fossil remnants from the early Proterozoic have been registered. In order to demineralize the skeletal structures of the glass sponge Hyalonema sieboldi, we have used for the first time a strategy of slow leaching of the silicon-bearing component, based on the usage of alkaline solutions of sodium hydroxide, sodium dodecyl sulfate, and an anionic biosurfactant of a rhamnolipid nature. The obtained data unequivocally corroborate the presence of a fibrillar protein matrix functioning as a basis for silicon biomineralization in the basal spicules of H. sieboldi. Also, it has been found for the first time that the protein matrix is constructed of a collagenous protein. The technical approach proposed here might appear important for the study of the structural organization of skeletons in other silicon-bearing animals and, in an applied aspect, to work out new biomaterials for implantology and biocomposites, in order to use the latter as bioactive additives.