The Loss of Structural Integrity of 3D Chitin Scaffolds from Aplysina aerophoba Marine Demosponge after Treatment with LiOH.

Aminopolysaccharide chitin is one of the main structural biopolymers in sponges that is responsible for the mechanical stability of their unique 3D-structured microfibrous and porous skeletons. Chitin in representatives of exclusively marine Verongiida demosponges exists in the form of biocomposite-based scaffolds chemically bounded with biominerals, lipids, proteins, and bromotyrosines. Treatment with alkalis remains one of the classical approaches to isolate pure chitin from the sponge skeleton. For the first time, we carried out extraction of multilayered, tube-like chitin from skeletons of cultivated Aplysina aerophoba demosponge using 1% LiOH solution at 65 °C following sonication. Surprisingly, this approach leads not only to the isolation of chitinous scaffolds but also to their dissolution and the formation of amorphous-like matter. Simultaneously, isofistularin-containing extracts have been obtained. Due to the absence of any changes between the chitin standard derived from arthropods and the sponge-derived chitin treated with LiOH under the same experimental conditions, we suggest that bromotyrosines in A. aerophoba sponge represent the target for lithium ion activity with respect to the formation of LiBr. This compound, however, is a well-recognized solubilizing reagent of diverse biopolymers including cellulose and chitosan. We propose a possible dissolution mechanism of this very special kind of sponge chitin.

Evaluation of a protocol to assess a novel artificial biofilm equivalent for dentures-A prospective clinical pilot study.

OBJECTIVES: This pilot study aimed to carry out preliminary tests of the removability of an artificial biofilm equivalent (ABE) and to verify the reproducibility of the ABE testing protocol for a planned main study. BACKGROUND: There is a lack of data to develop suitable artificial biofilm substitutes, which may be helpful to perform denture hygiene education and to carry out in vitro examinations of oral hygiene products. MATERIALS AND METHODS: This single-group, prospective, longitudinal, interventional pilot study was conducted in Dresden (Germany) from February until December 2020. Participants were recruited who wore fully functional upper complete dentures. Denture biofilm was grown on acrylic specimens by wearing dentures for 12 h and 36 h using intraoral appliances. Acrylic specimens were coated with ABEs of three compositions: chitosan (ChS) 0.3 g, methylcellulose (MC) 1.7 g; ChS 0.2 g, MC 1.8 g; ChS 0.1 g, MC 1.9 g (labelled 1.7MC, 1.8MC and 1.9MC, respectively). All specimens underwent standardised mechanical brushing. The percentages of remaining biofilm (POB) were measured. RESULTS: Thirty-one participants were prescreened, and eight (26%) were included. The appliances were well tolerated, and biofilm was collected. ABE was prepared and brushed as planned. Three and six brushing strokes were needed to remove 12-h and 36-h natural denture biofilm, respectively. Correspondingly, three brushing strokes were needed to remove 1.9MC ABE and six brushing strokes to remove 1.8MC and 1.7MC ABE. A reproducibility of ABE removal was indicated. CONCLUSION: The removability of ABE and the ABE testing protocol were feasible and reproducible for conducting the future main study.

Electrolysis as a Universal Approach for Isolation of Diverse Chitin Scaffolds from Selected Marine Demosponges.

Three-dimensional chitinous scaffolds often used in regenerative medicine, tissue engineering, biomimetics and technology are mostly isolated from marine organisms, such as marine sponges (Porifera). In this work, we report the results of the electrochemical isolation of the ready to use chitinous matrices from three species of verongiid demosponges (Aplysina archeri, Ianthella basta and Suberea clavata) as a perfect example of possible morphological and chemical dimorphism in the case of the marine chitin sources. The electrolysis of concentrated Na2SO4 aqueous solution showed its superiority over the chemical chitin isolation method in terms of the treatment time reduction: only 5.5 h for A. archeri, 16.5 h for I. basta and 20 h for the S. clavata sample. Further investigation of the isolated scaffolds by digital microscopy and SEM showed that the electrolysis-supported isolation process obtains chitinous scaffolds with well-preserved spatial structure and it can be competitive to other alternative chitin isolation techniques that use external accelerating factors such as microwave irradiation or atmospheric plasma. Moreover, the infrared spectroscopy (ATR-FTIR) proved that with the applied electrochemical conditions, the transformation into chitosan does not take place.

Functionalization of 3D Chitinous Skeletal Scaffolds of Sponge Origin Using Silver Nanoparticles and Their Antibacterial Properties.

Chitin, as one of nature's most abundant structural polysaccharides, possesses worldwide, high industrial potential and a functionality that is topically pertinent. Nowadays, the metallization of naturally predesigned, 3D chitinous scaffolds originating from marine sponges is drawing focused attention. These invertebrates represent a unique, renewable source of specialized chitin due to their ability to grow under marine farming conditions. In this study, the development of composite material in the form of 3D chitin-based skeletal scaffolds covered with silver nanoparticles (AgNPs) and Ag-bromide is described for the first time. Additionally, the antibacterial properties of the obtained materials and their possible applications as a water filtration system are also investigated.

Naturally pre-designed biomaterials: Spider molting cuticle as a functional crude oil sorbent.

Diverse fields of modern environmental technology are nowadays focused on the discovery and development of new sources for oil spill removal. An especially interesting type of sorbents is those of natural origin-biosorbents-as ready-to-use constructs with biodegradable, nontoxic, renewable and cost-efficient properties. Moreover, the growing problem of microplastic-related contamination in the oceans further encourages the use of biosorbents. Here, for the first time, naturally pre-designed molting cuticles of the Theraphosidae spider Avicularia sp. "Peru purple", as part of constituting a large-scale spider origin waste material, were used for efficient sorption of crude oil. Compared with currently used materials, the proposed biosorbent of spider cuticular origin demonstrates excellent ability to remain on the water surface for a long time. In this study the morphology and hydrophobic features of Theraphosidae cuticle are investigated for the first time. The unique surface morphology and very low surface free energy (4.47 ± 0.08 mN/m) give the cuticle-based, tube-like, porous biosorbent excellent oleophilic-hydrophobic properties. The crude oil sorption capacities of A. sp. "Peru purple" molt structures in sea water, distilled water and fresh water were measured at 12.6 g/g, 15.8 g/g and 16.6 g/g respectively. These results indicate that this biomaterial is more efficient than such currently used fibrous sorbents as human hairs or chicken feathers. Four cycles of desorption were performed and confirmed the reusability of the proposed biosorbent. We suggest that the oil adsorption mechanism is related to the brush-like and microporous structure of the tubular spider molting cuticles and may also involve interaction between the cuticular wax layers and crude oil.

Biomechanical Evaluation of Mandibular Condyle Fracture Osteosynthesis Using the Rhombic Three-Dimensional Condylar Fracture Plate.

PURPOSE: The aim of the present study was to evaluate the biomechanical stability of osteosynthesis in mandibular condyle fractures using a newly designed rhombic 3-dimensional (3D) condylar fracture plate and compare it with that using standard two 4-hole miniplates and with that in nonfractured condyles. MATERIALS AND METHODS: Using 200 porcine mandibles, 3 different monocortical plating techniques were evaluated. The condyles were fractured along a defined line tangentially through the sigmoid notch and perpendicular to the posterior border. After anatomic reduction, osteosynthesis was performed using either standard rhombic 3D condylar fracture plates and standard screws (group A) or locking rhombic 3D condylar fracture plates, which were fixed either with standard screws (group B) or locking screws (group C). For comparison, nonfractured condyles (group D) and condyles fixed with standard two 4-hole miniplates and 8 screws (group E) were included. Using a universal mechanical testing machine (TIRA Test 2720; TIRA GmbH Schalkau, Germany), each group was subjected to linear loading from laterally to medially, medially to laterally, anteriorly to posteriorly, and posteriorly to anteriorly. The maximum axial force and displacement at the maximum force were measured. The mean values were compared for statistical significance using analysis of variance with Bonferroni's correction (statistical significance set at P < .05). RESULTS: The main mode of failure in the plating techniques investigated was the pull out of screws from the proximal fragment. We found no statistically significant differences in the stability of osteosynthesis between the two 4-hole miniplates and the rhombic 3D condylar fracture plate when loading from posteriorly to anteriorly, laterally to medially, and medially to laterally. However, when loading from anteriorly to posteriorly, a statistically significant difference between the standard and locking system and the two 4-hole miniplate system was observed, with the latter proving more stable. CONCLUSIONS: The results of the present biomechanical study suggest that the rhombic 3D condylar fracture plates are suitable for the treatment of condylar neck fractures. Both types of the plate are able to resist physiologic strains comparable to the two 4-hole miniplates.

New Source of 3D Chitin Scaffolds: The Red Sea Demosponge Pseudoceratina arabica (Pseudoceratinidae, Verongiida).

The bioactive bromotyrosine-derived alkaloids and unique morphologically-defined fibrous skeleton of chitin origin have been found recently in marine demosponges of the order Verongiida. The sophisticated three-dimensional (3D) structure of skeletal chitinous scaffolds supported their use in biomedicine, tissue engineering as well as in diverse modern technologies. The goal of this study was the screening of new species of the order Verongiida to find another renewable source of naturally prefabricated 3D chitinous scaffolds. Special attention was paid to demosponge species, which could be farmed on large scale using marine aquaculture methods. In this study, the demosponge Pseudoceratina arabica collected in the coastal waters of the Egyptian Red Sea was examined as a potential source of chitin for the first time. Various bioanalytical tools including scanning electron microscopy (SEM), fluorescence microscopy, FTIR analysis, Calcofluor white staining, electrospray ionization mass spectrometry (ESI-MS), as well as a chitinase digestion assay were successfully used to confirm the discovery of α-chitin within the skeleton of P. arabica. The current finding should make an important contribution to the field of application of this verongiid sponge as a novel renewable source of biologically-active metabolites and chitin, which are important for development of the blue biotechnology especially in marine oriented biomedicine.

Translucency of Zirconia Ceramics before and after Artificial Aging.

PURPOSE: The aging of zirconia ceramics (Y-TZP) is associated with tetragonal to monoclinic phase transformation. This change in microstructure may affect the optical properties of the ceramic. This study examines the effect of aging on the translucency of different zirconia materials. MATERIALS AND METHODS: 120 disc-shaped specimens were fabricated from four zirconia materials: Cercon ht white, BruxZir Solid Zirconia, Zenostar T0, Lava Plus (n = 30 per group). Accelerated aging was performed in a steam autoclave (134°C, 0.2 MPa, 5 hours). CIELab coordinates (L*, a*, b*) and luminous reflectance (Y) were measured with a spectrophotometer before and after aging. Contrast ratio (CR) and translucency parameter (TP) were calculated from the L*, a*, b*, and Y tristimulus values. The general linear model (Bonferroni adjusted) was used to compare both parameters before and after aging, as well as between the different zirconia materials (p ≤ 0.05). RESULTS: CR and TP differed significantly before and after aging in all groups tested. Before aging, Zenostar T showed the highest and Lava Plus showed the lowest translucency. After aging, Cercon ht and Zenostar T showed the highest and BruxZir and Lava Plus the lowest translucency. CONCLUSIONS: Aging reduced the translucency in all specimens tested. Furthermore, translucency differed between the zirconia brands tested. Nevertheless, the differences were below the detectability threshold of the human eye. The aging process can influence the translucency and thus the esthetic outcome of zirconia restorations; however, the changes in translucency were minimal and probably undetectable by the human eye.

First Report on Chitin in a Non-Verongiid Marine Demosponge: The Mycale euplectellioides Case.

Sponges (Porifera) are recognized as aquatic multicellular organisms which developed an effective biochemical pathway over millions of years of evolution to produce both biologically active secondary metabolites and biopolymer-based skeletal structures. Among marine demosponges, only representatives of the Verongiida order are known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. The unique three-dimensional (3D) architecture of such chitinous skeletons opens the widow for their recent applications as adsorbents, as well as scaffolds for tissue engineering and biomimetics. This study has the ambitious goal of monitoring other orders beyond Verongiida demosponges and finding alternative sources of naturally prestructured chitinous scaffolds; especially in those demosponge species which can be cultivated at large scales using marine farming conditions. Special attention has been paid to the demosponge Mycale euplectellioides(Heteroscleromorpha: Poecilosclerida: Mycalidae) collected in the Red Sea. For the first time, we present here a detailed study of the isolation of chitin from the skeleton of this sponge, as well as its identification using diverse bioanalytical tools. Calcofluor white staining, Fourier-transform Infrared Spcetcroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS), scanning electron microscopy (SEM), and fluorescence microscopy, as well as a chitinase digestion assay were applied in order to confirm with strong evidence the finding of a-chitin in the skeleton of M. euplectellioides. We suggest that the discovery of chitin within representatives of the Mycale genus is a promising step in their evaluation of these globally distributed sponges as new renewable sources for both biologically active metabolites and chitin, which are of prospective use for pharmacology and biomaterials oriented biomedicine, respectively.

Neuropsychological Training of Attention Improves MS-Related Fatigue: Results of a Randomized, Placebo-Controlled, Double-Blind Pilot Study.

BACKGROUND: Attentional deficits may be pathophysiologically relevant in MS-associated fatigue. PATIENTS AND METHODS: Thirty MS patients with fatigue and attentional deficits in neuropsychological testing participated in this randomized, placebo-controlled, double-blind trial. The intervention group (IG; n = 14) was treated with 10 h of computerized, specific neuropsychological training performing simple reaction time tasks, whereas the control group (CG; n = 16) also runs through computerized, but unspecific neuropsychological training using tasks without time components. The subjective feeling of fatigue was assessed with the Würzburg Fatigue Inventory for Multiple Sclerosis (WEIMuS) questionnaire, and testing of alertness was used as an objective measure at baseline and after the 2-week study period. RESULTS: Reaction times of alertness were significantly decreased in IG but not CG after 2 weeks. The subjective feeling of fatigue was ameliorated in both groups but more pronounced in IG. Effect sizes were below 0.7 for alertness and WEIMuS scores in CG but large and clinically meaningful in IG for both measures. CONCLUSIONS: Our pilot study suggests that neuropsychological training of attention may improve both measures of fatigue. The parallel improvement of attentional deficits and subjective fatigue after specific neuropsychological training support previous findings that fatigue may be at least partially caused by impaired intensity of attention.

Development and first data of a customized short tracheal cannula based on digital data.

PURPOSE: At the moment, there is an inadequate margin fit of commercially available stoma buttons. The aim of the present study was to develop a customized short tracheal cannula based on digital data. Furthermore, the applied material has to be evaluated considering germ colonization and appropriate cleaning procedures. METHODS: Computed tomographies of 53 patients who underwent laryngectomy were surveyed. Based on the digital data, a customized short tracheal cannula was created and manufactured from silicone. The new cannula was incorporated in ten patients and worn for 4 weeks. A clinical examination of an otolaryngologist and subjective assessment of the patients were carried out. Furthermore, microbiological test considering germ colonization was performed. RESULTS: The customized short tracheal cannula could be incorporated in all patients. The clinical results showed no irritation or mucosal lesions. The subjective individual evaluation by the patients was promising. The proposals for improvement could be considered. The microbiological examination revealed a higher contamination of the silicone compared to the silver cannulas. Both chemical and mechanical decontamination showed sufficient results. CONCLUSION: A workflow for development and manufacturing of a customized short tracheal cannula from digital data could be established. The cannula is compatible to standard equipment and routine cleaning procedures. Clinical studies are required to evaluate the potential benefit for patients.

Creation and utilization of a digital database for nasal prosthesis models.

BACKGROUND: The study describes the development and implementation of a digital nose database in order to provide patients with nasal prostheses following rhinectomy. Mirrored data for computer-aided design (CAD) cannot be used due to the unpaired structure of the nose. MATERIALS AND METHODS: The faces of 202 people were digitized using a 3-dimension (3D) scanner. The noses were scaled, measured and classified according to objective criteria. The physician, the patient and the anaplastologist can collaborate in order to select an appropriate nose from the multitude of existing nose types and sizes. Virtual 'fittings' and an individual adaptation of the nose are feasible. For this purpose the epiTecture software was applied. The selected nose is then created on a 3D printer as a thermopolymer model. This model can be fitted and corrected as a physical model on the patient. The remaining steps are identical to conventional prosthesis production. RESULTS: A digital nose database was developed at the University Hospital Dresden with the help of the epiTecture software. Instructions for usage are illustrated using the example of a patient. CONCLUSIONS: The process of providing nasal prostheses described in this paper is different from conventional processes. This is primarily due to the elimination of physical modeling, causing substantially less strain for the patient.

Retention of different temporary cements tested on zirconia crowns and titanium abutments in vitro.

PURPOSE: The aim of the present study was to examine the retention force of monolithic zirconia copings cemented with various temporary cements on implant abutments in vitro. METHODS: Sixty exercise implants with pre-screwed implant abutments were embedded in resin. Subsequently, 60 CAD/CAM manufactured zirconia copings were divided into three main groups [Harvard Implant Semi-permanent (HAV), implantlink semi Forte (IMP), Temp Bond NE (TBNE)]. The zirconia copings were cemented on the implant abutments and loaded with 35 N. Specimens were stored in distilled water (37 °C) for 24 h. Half of the test specimens of each group were subjected to a thermocycling (TC) process. Retention force was measured in a universal testing machine. Using magnifying glasses, the fracture mode was determined. Statistical analysis was performed applying the Kruskal-Wallis test, the post hoc test according to Dunn-Bonferroni and a chi-square test of independence. RESULTS: Without TC, IMP showed the highest retention of the three temporary luting agents (100.5 ± 39.14 N). The measured retention forces of IMP were higher than those of HAV (45.78 ± 15.66 N) and TBNE (61.16 ± 20.19 N). After TC, retention was reduced. IMP showed the greatest retentive strength (21.69 ± 13.61 N, three fail outs). HAV and TBNE showed pull-off forces of similar magnitude (17.38 ± 12.77 N and 16.97 ± 12.36 N, two fail outs). The fracture mode analysis showed different results regarding the tested cements before and after TC (facture type before/after TC): IMP (III+II/III), HAV (I/II) and TBNE (III/III). There were clear differences of the fracture modes regarding the examination before and after TC. CONCLUSIONS: Within the limits of this study, IMP showed the highest pull-off forces under the chosen test conditions. All three temporary luting agents showed lower retention forces after TC. Retention values in the individual cement classes were very heterogeneous. Easy cement removal in the crown lumen favours the dominance of adhesive cement fractures on the abutment and adhesive/cohesive cement fractures on the abutment with HAV appears advantageous in case of recementation of the superstructure.

Adapting the Pore Size of Individual, 3D-Printed CPC Scaffolds in Maxillofacial Surgery.

Three dimensional (3D) printing allows additive manufacturing of patient specific scaffolds with varying pore size and geometry. Such porous scaffolds, made of 3D-printable bone-like calcium phosphate cement (CPC), are suitable for bone augmentation due to their benefit for osteogenesis. Their pores allow blood-, bone- and stem cells to migrate, colonize and finally integrate into the adjacent tissue. Furthermore, the pore size affects the scaffold's stability. Since scaffolds in maxillofacial surgery have to withstand high forces within the jaw, adequate mechanical properties are of high clinical importance. Although many studies have investigated CPC for bone augmentation, the ideal porosity for specific indications has not been defined yet. We investigated 3D printed CPC cubes with increasing pore sizes and different printing orientations regarding cell migration and mechanical properties in comparison to commercially available bone substitutes. Furthermore, by investigating clinical cases, the scaffolds' designs were adapted to resemble the in vivo conditions as accurately as possible. Our findings suggest that the pore size of CPC scaffolds for bone augmentation in maxillofacial surgery necessarily needs to be adapted to the surgical site. Scaffolds for sites that are not exposed to high forces, such as the sinus floor, should be printed with a pore size of 750 µm to benefit from enhanced cell infiltration. In contrast, for areas exposed to high pressures, such as the lateral mandible, scaffolds should be manufactured with a pore size of 490 µm to guarantee adequate cell migration and in order to withstand the high forces during the chewing process.

Mechanical and Surface Properties of Resilient Denture Liners Modified with Chitosan Salts.

Chitosan (CS) and its derivatives show antibacterial and antifungal properties and could help treat and prevent denture stomatitis (DS). Mechanical and surface properties of resilient denture liners were evaluated when modified with CS salts. CS-hydrochloride (CS-HCl) and CS-glutamate (CS-G) were added to resilient denture liners Ufi Gel P and Coe-Soft at four different concentrations (0.1%, 0.2%, 0.4%, 1% w/w) from which specimens were produced, as well as a control group of each material with no added CS salt. Ten specimens per group (Ø 35 mm, height 6 mm) were manufactured. They were stored in distilled water at 37 °C for a total of 30 days (d). Shore A hardness (SHA) and surface roughness (Ra) were evaluated after 24 h (T1), 7 d (T2), 14 d (T3) and 30 d (T4). Kruskal-Wallis and U-test (Bonferroni-Holm adjusted) were used for statistical analysis (p ≤ 0.05). Ra increased significantly once CS salts were added. SHA increased significantly for some groups, but all specimens fulfilled requirements set by ISO 10139-2:2016. Modification with CS salts does not influence the mechanical properties of the modified resilient denture liners in a clinically relevant manner. Despite the increased roughness, the concept is suitable for further studies. Especially antimicrobial/antibiofilm studies are needed.

The Influence of Thrust Force on the Vitality of Bone Chips Harvested for Autologous Augmentation during Dental Implantation.

Bone drill chips that are collected during implant site preparation can be reused as autologous bone-grafting material for alveolar ridge augmentation. This study characterized five market-leading implant drill sets regarding their geometric properties and ability to produce vital bone chips. The drill geometry of each tool of five commercial implant drill sets was characterized while using optical profile projector devices and SEM. Bone chips were collected during the in vitro preparation of porcine jaw bone with the various drill sets. Produced bone chip masses were measured. The bone chips were cultured in vitro and the number of outgrown cells was determined and measurand for vitality. Furthermore, the thrust force and cutting torque were recorded to examine the mechanical loads of the manual drilling process. The tool geometry and set configuration of one out of five implant drill sets appears to be superior regarding chip mass, vitality, and thrust force. It could be proven that there is a correlation between vitality and thrust force. The thrust force is influenced by the cutting behavior of the tool, which in turn depends on the geometry of the tool. The tool geometry has an influence on the vitality of the augmentation material due to this relationship.

Spider Chitin. The biomimetic potential and applications of Caribena versicolor tubular chitin.

Diverse fields of modern technology and biomedicine can benefit from the application of ready-to-use chitin-based scaffolds. In this work we show for the first time the applicability of tubular and porous chitin from Caribena versicolor spiders as a scaffold for the development of an effective CuO/Cu(OH)2 catalyst for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AM), and as a scaffold for the tissue engineering of selected cells. The formation of CuO/Cu(OH)2 phases on and within the chitinous tubes leads to a hybrid material with excellent catalytic performance with respect to the reduction of p-nitrophenol. On the other hand, experimental results provide for the first time strong evidence for the biocompatibility of spider chitin with different cell types, a human progenitor cell line (hPheo1), as well as cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) that were cultured on a tube-like scaffold.

Extreme biomimetics: Preservation of molecular detail in centimeter-scale samples of biological meshes laid down by sponges.

Fabrication of biomimetic materials and scaffolds is usually a micro- or even nanoscale process; however, most testing and all manufacturing require larger-scale synthesis of nanoscale features. Here, we propose the utilization of naturally prefabricated three-dimensional (3D) spongin scaffolds that preserve molecular detail across centimeter-scale samples. The fine-scale structure of this collagenous resource is stable at temperatures of up to 1200°C and can produce up to 4 × 10-cm-large 3D microfibrous and nanoporous turbostratic graphite. Our findings highlight the fact that this turbostratic graphite is exceptional at preserving the nanostructural features typical for triple-helix collagen. The resulting carbon sponge resembles the shape and unique microarchitecture of the original spongin scaffold. Copper electroplating of the obtained composite leads to a hybrid material with excellent catalytic performance with respect to the reduction of p-nitrophenol in both freshwater and marine environments.

The demosponge Pseudoceratina purpurea as a new source of fibrous chitin.

Among marine demosponges (Porifera: Demospongiae), only representatives of the order Verongiida have been recognized to synthetize both biologically active substances as well as scaffolds-like fibrous skeletons made of structural aminopolysaccharide chitin. The unique 3D architecture of such scaffolds open perspectives for their applications in waste treatment, biomimetics and tissue engineering. Here, we focus special attention to the demosponge Pseudoceratina purpurea collected in the coastal waters of Singapore. For the first time the detailed description of the isolation of chitin from the skeleton of this sponge and its identification using diverse bioanalytical tools were carried out. Calcofluor white staining, FTIR analysis, electrospray ionization mass spectrometry (ESI-MS), SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm with strong evidence the finding of alpha-chitin in the skeleton of P. purpurea. We suggest that the discovery of chitin within representatives of Pseudoceratinidae family is a perspective step in evaluation of these verongiid sponges as novel renewable sources for both chitin and biologically active metabolites, which are of prospective use for marine oriented biomedicine and pharmacology, respectively.

Discovery of chitin in skeletons of non-verongiid Red Sea demosponges.

Marine demosponges (Porifera: Demospongiae) are recognized as first metazoans which have developed over millions of years of evolution effective survival strategies based on unique metabolic pathways to produce both biologically active secondary metabolites and biopolymer-based stiff skeletons with 3D architecture. Up to date, among marine demosponges, only representatives of the Verongiida order have been known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within skeletons of non-verongiid demosponges Acarnus wolffgangi and Echinoclathria gibbosa collected in the Red Sea. Calcofluor white staining, FTIR and Raman analysis, ESI-MS, SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm, with strong evidence, the finding of α-chitin in the skeleton of both species. We suggest that, the finding of chitin within these representatives of Poecilosclerida order is a promising step in the evaluation of these sponges as novel renewable sources for both biologically active metabolites and chitin, which are of prospective application for pharmacology and biomedicine.