POZNAN UNIVERSITY OF TECHNOLOGY FACULTY OF CHEMICAL TECHNOLOGY
INSTITUTE OF CHEMICAL TECHNOLOGY AND ENGINEERING
Ph.D. THESIS
Hydrothermal synthesis of selected inorganic compounds using spongin-based scaffolds
by
Tomasz Piotr Szatkowski, M.Sc., Eng.
Thesis submitted for the degree of Doctor of Philosophy
Specialty: Chemical Technology
Supervisor: Professor TEOFIL JESIONOWSKI (Poland) Co-supervisor: Professor HERMANN EHRLICH (Germany)
Poznan 2017
Projekt POKL.04.01.01-00-049/13
Rozwój kształcenia w zakresie nanotechnologii na Politechnice Poznańskiej w oparciu o współpracę z Centrum NanoBioMedycznym UAM
i Universita degliStudi di Trieste
This work was financially supported by European grant POKL.04.01.01-00-049/13,
“Developing education in the field of nanotechnology at Poznan University of Technology based on cooperation with the Faculty of Physics PUT, NanoBioMedical Centre AMU, Faculty of Chemistry AMU and Universita degli Studi di Trieste”.
Thesis was prepared partially within the framework of Research Grant for Doctoral Candidates and Young Academics and Scientists, supported by DAAD – German Academic Exchange Service, no 323-A/12/84687.
Acknowledgements
I would first like to thank my thesis supervisors, Professor Teofil Jesionowski and Professor Hermann Ehrlich whose assistance and guidance cannot be overestimated.
Thank you that the doors to your offices were always open for me whenever I had questions, doubts or needed directions.
I would also like to express my sincere gratitude to my academic friends:
Dr Marcin Wysokowski, Dr Łukasz Klapiszewski, and B.Sc. Andre Ehrlich.
You have always assisted me when help was needed, and advised me when I sought for knowledge and expertise.
Thank to you these 5 years were not always a tough work.
At this point I am also very grateful to all my scientific teammates for cooperation and scientific discussions we had.
Last but not least, I would like to express the deepest appreciation to my whole family (the native one as well as the acquired during my Ph.D.), and to my wife in particular,
for their patience and support since the beginning of the doctoral course.
Table of contents
1 Introduction ... 8
2 Sponges (Porifera) as unique source for bioinspired materials science ... 11
2.1 General information about sponges and their skeletal structures ... 11
2.2 Comparison of chitin and spongin in demosponges ... 17
2.3 Chemistry of structural protein spongin ... 19
2.4 Structural properties of spongin–based skeletons... 25
2.5 Spongin as biomaterial – from historical applications to tissue engineering ... 29
2.6 Thermal stability of spongin and other proteins... 34
3 Hydrothermal synthesis and Extreme Biomimetics strategy ... 39
3.1 Foreword ... 39
3.2 Inspirations for Extreme Biomimetics ... 40
3.3 Hydrothermal synthesis of metal oxides and organic-inorganic composites ... 43
4 Aim of the work ... 50
5 Materials and methods ... 51
5.1 Isolation of spongin ... 52
5.2 Hydrothermal silicification of spongin ... 53
5.2.1 Silicification of spongin fibers via base catalyzed sol-gel reaction ... 53
5.2.2 Silicification of spongin fibers via acid catalyzed sol-gel process ... 53
5.2.3 Synthesis of spongin templated quartz particles using hydrothermal treatment ... 54
5.3 Hydrothermal synthesis of spongin-calcium phosphate composite ... 54
5.4 Nanostructured hematite-spongin composite obtained using Extreme Biomimetic approach ... 55
5.5 Hydrothermally treated spongin-titania composite ... 55
5.6 Carbonized spongin scaffold as a support for manganese(IV) oxide ... 56
5.7 Characterization techniques ... 56
5.7.1 Fourier-transform infrared spectroscopy (FTIR) ... 56
5.7.2 Raman spectroscopy ... 56
5.7.3 X-ray photoelectron spectroscopy (XPS) ... 57
5.7.4 Energy dispersive X-ray spectroscopy (EDS) ... 57
5.7.5 X-ray diffraction spectroscopy (XRD) ... 57
5.7.6 Elemental analysis (EA)... 57
5.7.7 Scanning electron microscopy (SEM) ... 58
5.7.8 Transmission electron microscopy (TEM) ... 58
5.7.9 Thermal gravimetry (TG) and differential scanning calorimetry (DSC) ... 58
5.7.10 Particle size distribution via non-invasive backscattering method (NIBS) ... 58
5.7.11 Low-temperature nitrogen sorption ... 58
5.7.12 Mercury intrusion porosimetry ... 59
5.7.13 Mechanical measurements ... 59
5.7.14 Electrochemical measurements ... 59
5.7.15 Cytotoxicity study ... 60
5.7.16 Estimation of proliferation rate of MG63 cells seeded on spongin scaffolds ... 60
6 Physicochemical properties of spongin ... 62
6.1 Introduction ... 62
6.2 Structural properties of spongin ... 62
6.3 Physicochemical properties of spongin ... 64
6.4 Conclusions ... 67
7 Hydrothermal silicification of spongin ... 68
7.1 Introduction ... 68
7.2 Structural properties of hydrothermally silicified spongin ... 70
7.3 Physicochemical properties of silicified spongin ... 71
7.4 Synthesis of spongin templated quartz particles using hydrothermal treatment ... 73
7.5 Conclusions ... 77
8 Hydrothermal synthesis of spongin-calcium phosphate composite ... 78
8.1 Introduction ... 78
8.2 Structural and physicochemical properties of the hydrothermally treated calcium phosphate .. 79
8.3 Structural properties of the hydrothermally prepared spongin-CP composite ... 84
8.4 Mechanical performance spongin-CP composite ... 86
8.5 Biological activity of spongin-CP composite ... 87
8.5.1 Cytotoxicity of extracts obtained with use of spongin and spongin-CP composite ... 87
8.5.2 Proliferation rate of MG63 cells on sponge skeleton and spongin-CP composite in vitro . 90 8.6 Conclusions ... 91
9 Nanostructured hematite-spongin composite obtained using Extreme Biomimetic approach... 93
9.1 Introduction ... 93
9.2 Structural properties of α-Fe2O3-spongin composite ... 94
9.3 Physicochemical properties of α-Fe2O3–spongin composite ... 95
9.4 Electrochemical application of α-Fe2O3–spongin composite ... 100
9.5 Conclusions ... 101
10 Spongin-titanium(IV) oxide for removal of C.I. Basic Blue 9 from aqueous solution ... 102
10.1 Introduction ... 102
10.2 Preliminary tests: selection of optimum temperature of synthesis ... 103
10.3 Structural properties of spongin-titanium(IV) oxide composite obtained at 120 °C ... 106
10.4 Photodegradation of C.I. Basic Blue 9 dye using spongin – titanium(IV) oxide composite .... 112
10.5 Conclusion ... 113
11 Carbonized skeleton of Demosponge as a support for manganese(IV) oxide ... 114
11.1 Introduction ... 114
11.2 Structural properties of carbon sponge-MnO2 composite (CS/MnO2) ... 116
11.3 Physicochemical properties of CS/MnO2 composite... 117
11.4 Cyclic voltammetry and galvanostatic charge–discharge measurements ... 123
11.5 Conclusions ... 125
12 Summary and Outlook ... 126
References ... 129
Abstract ... 164
Streszczenie... 167
Scientific activity ... 170
Chapter 1
Introduction
Living organism that we can observe today are the result of about 3.8 billion years of evolution. During that time, Nature has been experimenting by trials and errors with broad variety of commonly found materials in order to find solutions to various challenges, such as collection of food, defense against predators, reproduction, etc.
Nature eventually developed highly efficient substances and objects designed and adjusted for specific biological needs and environmental conditions. These biological materials are characterized with remarkable performance. Often, they are structurally organized from the molecular to the nano-, micro- and macroscale in a hierarchical manner, creating intricate architectural designs that ultimately makes up a myriad of different functional elements.1
It is not surprising that humans very early learned to observe and mimic the surrounding environment. For instance the Chinese tried to make artificial silk about 3000 years ago, and Leonardo da Vinci studied the way birds fly and came out with designs for his flying machines.2 However, the concept to mimic Nature is relatively new, and the term biomimetics was proposed for the first time in 1969 by Schmitt as a field of study copying, imitating and learning from biology.3 Since then, the trend grew to a separate, very popular field of science interconnecting principles of physics, chemistry, mechanical engineering, materials science, mobility control, sensors, and many other fields.4 Interestingly, with advancement of scientific tools and techniques it was possible to study the natural mechanisms occurring at nano- and microlevels.
One of the most intriguing aspects of biomimetics is the formation of biominerals- containing composite materials based on organic templates. Biomineralization gets enormous attention (based on the Scopus database search analysis, until June 2017 more than 12,100 scores appeared containing phrase “biomineralization” and the trend is growing). Biomineralization is a discipline located at the interface of earth and life bringing cross-disciplinary training and new experimental and computational methods to the most daunting problems. It can be divided into two distinctive groups according to the influence of biological systems, and so, it can be either “biologically induced” or
“biologically controlled”.5 In this process, organisms typically accumulate the precursors (e.g. ions of metals) required to synthesize biominerals from their environment, and create composites of organic macromolecules (e.g. proteins, polysaccharides) and inorganic compounds which can be characterized with distinctive structural and mechanical properties.6
What is fascinating, biomineralization occurs also in extreme environmental niches, at temperatures higher than 100 °C around hydrothermal vents, geothermal pipelines, and hot springs, as well as at freezing waters of Arctic and Antarctic seas at
temperatures from -1.9 °C to 4 °C (see for review 7). Here, organisms known as polyextremophiles represent the sources for bioinspiration. Very recently the investigations concerning these stunning phenomena were grouped under the term Extreme Biomimetics.6,7
In contrast to traditional aspects of biomimetic synthesis of organic-inorganic materials at ambient temperatures, the goal of Extreme Biomimetics is to bring together broad variety of extreme (from the biological point of view) chemical reactions with templates of biological origin, and to develop the next generation of hybrid composites with enhanced properties.7 The idea of Extreme Biomimetics was born at the crossroads between such scientific disciplines as Prebiotic Chemistry and Mineralogy, Astrobiology, Evolutionary Biology, Hydrothermal Chemistry and Biochemistry, and Exobiology. These in turn include scientific directions such as the Primordial Soup Theory, the Origin and Evolution of Life, and Extreme Biomineralization (Figure 1).7
Thus, the basic principle of this concept is to exploit biopolymers that are chemically and thermally stable under these very specific conditions in vitro. So far several biopolymers were identified as thermally stable enough to serve as an organic template in reactions carried out using Extreme Biomimetics approach, that includes chitin and chitosan,8 cellulose,9 and silk.10,11 These biological materials were used as basis for synthesis of novel nanostructured composites with stunning properties and functionality, such as chitin hydrogels,12 cellulose shape controlled TiO2 nanoparticles for photocatalytic applications,13 or silk as source for synthesis of nitrogen-doped carbon dots useful in bioimaging.14
In this thesis it is stated for the first time that spongin, a structural protein originating from marine demosponges which possesses high thermostability (up to 360 °C) can be used as novel and renewable template for broad variety of hydrothermal synthesis reactions with respect to silica as well as other selected oxides using Extreme Biomimetics approach. Spongin represents naturally prestructured (“ready to use”) protein which can be isolated in the form of 3D centimeter large scaffolds. It is highly perspective even at industrial scale especially due to ability of demosponges to grow under marine farming conditions worldwide.15,16 The theoretical part of the dissertation provides not only the state-of-the-art knowledge on the use of spongin as an organic matrix with 3D architecture, but also refers to the first scientific annotation on demosponges, giving the reader a wide and historical view on the discussed topic.
Moreover, it refers to the new scientific direction of Extreme Biomimetics, presenting its principles, attractive features and potential applications.
The experimental part of this dissertations is concentrated on utilization of 3-dimensional spongin template, isolated from the skeleton of Hippospongia communis marine demosponge, as a microporous support for inorganic materials, mineralized under hydrothermal conditions. Hydrothermal synthesis serves as a tool for bringing natural phenomena occurring at extreme niches of the environment into laboratory
practice, allowing for synthesis of organic-inorganic materials. The selection of inorganic precursors is not accidental, but inspired by the natural occurrence of biomineralized skeletal constructs which are found in sponges.
Figure 1. Schematic view on the place of Extreme Biomimetics on the multidisciplinary crossroads (adapted from 7).
The methodology of experiments, as well as the applied analytical tools and technics are presented in details. For the first time spongin-based composite materials which contain silica, calcium-phosphate, hematite, titania and manganese oxide have been developed. The obtained results are accompanied with discussion with respect to physicochemical properties of the developed materials as well as their potential applications in biomedicine, electrochemistry and photocatalysis. The general conclusions are given in the final section of the thesis highlighting most important endpoints.
Chapter 2
Sponges (Porifera) as unique source for bioinspired materials science
Contents
2.1. General information about sponges and their skeletal structures 2.2. Comparison of chitin and spongin in demosponges
2.3. Chemistry of structural protein spongin
2.4. Structural properties of spongin–based skeletons
2.5. Spongin as biomaterial – from historical applications to tissue engineering 2.6. Thermal stability of spongin
2.1 General information about sponges and their skeletal structures
Sponges are included into animal kingdom, phylum Porifera (from Latin meaning
“pore bearer”). The first records of sponge-related sediments were found in petroleums and bitumens from Early Proterozoic (≈ 1.8 Bya) to Miocene (≈ 15 Mya) and contained 24-isopropylcholestanes, a novel group of C30 steroids produced by sponges, indicating a relative abundance of these organism already at that time.17 However, first sponge remains which possessed today’s morphological shape were found in the Early Vendian (Ediacaran) Doushantuo phosphate deposit in central Guizhou (South China), which has an age of approximately 580 Mya.18 The fauna indicates that animals lived 40 to 50 million years before climatic extremes and biological evolutionary developments, during the so called Cambrian Explosion, which occurred around 545 Myr and is graphically presented in Figure 2.18
Sponges are probably one of the earliest multicellular organisms (Metazoa)19 to diverge, however, since their emergence they underwent minor evolutionary change (Figure 3). This group of simple animals inhabits exclusively aquatic environments and is widely distributed among marine as well as freshwater systems. They thrive on hard as well as soft substrata, from tropical to polar latitudes, from deep to shallow waters, and are considered as one of the major groups of species in hard- bottom communities.20
Figure 2. Geological time scale showing the appearance of specific groups of life.
It can be noticed that sponges appeared prior to the Cambrian explosion.21
Sponges are generally sessile animals, which live attached to numerous substrata, including rocks, corals, mollusk shells, wood as well as artificial underwater constructs which are often used as a support for attachment of sponge fragments in cultivation farms. Exceptionally, some sponges can move on short distances using crawling-like mechanism, and are capable of traveling 1-4 mm per day.22 In order to retrieve food particles, like phytoplankton, bacteria, detritus micro algae, dead organic particles or other unicellular organisms (usually in range of 0.1 to 50 µm)23,24 sponges filter surrounding water with great efficiency. Due to unique design of the skeleton, some sponges are able to pump up to 24,000 liters of water per 1 kg of body per day.25
In order to perform pumping in an efficient manner sponges developed different levels of body organization (Figure 3).26 The simplest one is found in primitive sponges of the class Calcarea, where the body remains a simple tubular unit, reminding a vase with unfolded epithelial cells (“skin” cells) called ascon. The more complex grade of organization (sycon) occurs after folding of outer (pinacoderm) and inner (choanoderm) layer of cells resulting in a better developed outer surface area, thus enabling more efficient water filtration. The most complex organization of sponge body characterizes the leucon form. It appears after subdivision of flagellated surface into discrete spherical chambers (containing choanocyte cells responsible for water circulation).
Figure 3. Levels of sponge body organization indicating rising complexity of the body structure (adapted from 27).
Despite the general simplicity of the sponge body organization these organisms are built of several types of cells performing specific activity. Among them (listed and described in Table 1) only two are capable of forming of organ-like structures:
(i) pinacocytes, which construct an outer continuous layer, are responsible for separation of the sponge mesohyl from the environment, lining the external surface and creating the basal attachment; (ii) choanocytes, which are flagellated cells form chambers, and are responsible for creating pressure that drives the water current within the sponge body.26
Table 1. Names and primary function of cells composing sponges.26
Cells type Cells name Function
Cells of epithelial surfaces
Exopinacocytes form external layer; similar role to mammalian epithelium
Endopinacocytes line internal canals
Basopinacocytes form basal epithelium of the sponge (an attachment structure); an active secretion of the fibrillar collagen and polysaccharide complex
Porocytes components of the inhalant system; are able to control pores size
Choanocytes flagelled cells forming chambers and forcing water flow through canals of sponge
Skeleton creating cells Collencytes secrete fibrillary collagen, a fundamental component of the sponge skeleton working as a framework to entire sponge matrix
Lophocytes produce great amount of collagen primarily for basal attachment purpose
Spongocytes secrete spongin, a major binding element of the skeleton in several orders of Demospongiae
Sclerocytes mobile cells which contributes to formation of spicules
The histology of sponges (next to a reproductive characteristics, skeletal characteristics, consistency and shape, pigmentation, biochemistry, etc.) are one of the key parameters to classify the Porifera group which, is still not an easy task for many taxonomists due to instability and variability of the phylum. The biggest challenge to this day have been the systematics of the Demospongiae group, which accounts for about 83% of all sponges.28 The earliest noticeable work was the one of Topsent that gave basic arrangement for later works of others.29 The most comprehensive work, however, was published in 1953-1957 by Levi who for the first time incorporated reproductive characteristics for definition of subclass categories.30 The most recent classification divides the Porifera phylum into four classes:
Calcarea, Hexactinellida, Homoscleromorpha and Demospongiae.31 The characteristic of each group are briefly described in Table 2.
Table 2. Classification of sponges and typical characteristics of each class.
Class name Characteristics of the group
Calcarea exclusively marine;
skeleton entirely composed of calcium carbonate;
skeletal elements are not differentiated into megascleres and microscleres.
Hexactinellida often called “a glass sponge”;
exclusively marine, live in deep waters (from 200 m to more than 6000 m);
siliceous mineral skeleton composed of spicules with hexactinal structure (drawing attention of materials scientist);32
variable body shape.
Homoscleromorpha skeleton, if present, is composed of tetraxonic siliceous spicules with four equal rays (calthrops);
color varies from cream to blue, violet, green, yellow, deep brown, orange or red;
often found in dark or semi-dark ecosystems, generally located in shallow waters (some species have been found below 100 m).
Demospongiae largest and most diverse group; marine and fresh waters
siliceous spicules (either monaxonic or tetraxonic) and/or with a skeleton of spongin fibers or fibrillar collagen;
three orders (Dictyoceratida, Dendroceratida, and Verongida) are lacking siliceous spicules and known as keratose or horny sponges. Representatives of Verongida contain chitin.
As can be noticed from description in the Table 2, the skeleton is an integral and important part of sponges and plays a crucial role in their lives. Among Porifera one can observe vast diversity of the skeletal structures and for the construction they use materials of either organic or inorganic character. When inorganic it is either siliceous (amorphous SiO2), calcareous (CaCO3) or both. For instance, these two materials can be found combined in several species of Hexactinellida class (Caulophacus species)33 or demosponges of Verongida order.34 The siliceous skeleton is usually secreted by sclerocytes in form of complex needle-shape spicules (see Figure 4). These spicules often form a distinct skeleton (as in the fascinating group of Hexactinellida sponges which form a rigid skeleton), but occasionally they are loosely distributed throughout the sponge body without identifiable order or they are lacking entirely.35 If the siliceous skeleton is not secreted, as in the case of Dysidea avara demosponge, it is often replaced by incorporated extraneous materials like sand, rocks, or shells which ensure rigidity of the sponge body.
The organic skeleton was thought to be only of collageneous character until the work of Ehrlich et al.36 who as a first proved that also chitin, previously considered
as contamination, can be a main constituent of the fibrous skeleton of Verongida rigida,36 and as a structural template for biomineralization of silica in several species of glass sponges 37,38
Figure 4. SEM images showing the structural diversity of silica-based micro- and megascleres, not to scale, sizes vary between 0.01 mm to 1 mm (adapted from 28).
A very peculiar member of the collagen class family is spongin. This protein is found exclusively in Demospongiae, and will not be found in any other organism among animal kingdom. It plays a crucial role of a reinforcement loosely dispersed throughout the mesohyl, but occasionally it comprises the skeleton of demosponges.
In case of sponges of Dictyoceratida and Dendroceratida orders spongin can be a sole building material of structural elements, constructing 3D skeletal formations.
Besides the fact that spongin is a building material of skeleton of keratose sponges it plays also other functions in their organisms:26,39
• Spiculated spongin fibers – completely or partially bind the spicules together, which gives the animal enhanced elasticity and resistance. It can occur only at nodes of the spicules, acting as joints in vertebrates, or form a thick sheath around each spicule and nods;
• Shell of the gemmules – protection of asexual reproductive bodies formed within the tissues of sponges (occur mostly in freshwater sponges, covered with spherular shells and provide necessary mechanical protection);
• Spiculoids – spicule-like elements build of spongin, they are either free, or partly joined to the fibers of skeleton, strikingly similar to the inorganic spicules formed as diactines, triactines, or/and tetractines;
• Basal spongin – secreted by the basopinacocytes forms a more or less continuous layer allowing sponges to firmly attach to a substratum.
Regardless the function and location in some sponges, spongin is comprised of aligned microfibrils,40–42 about 10 nm in diameter which can be characterized with banding pattern and periodicity of about 60 nm,43 similarly to the characteristic periodicity of collagen (67 nm).44 Structural properties of spongin fibers are described in details in subchapter 2.4.
2.2 Comparison of chitin and spongin in demosponges
Chitin and spongin can be found in coexistence within a single sponge organism though they comprise two completely different structural materials, both at the angle of developmental and evolutional origin. As was already mentioned before the research published by Ehrlich et al.36 chitin was considered rather as an impurity than as an integral part of sponge skeleton. What is more, it appears that incorporation of chitin in spongin skeleton is not accidental because the resulting material is more rigid for the sake of elasticity, and acquires higher resistance to pressure and chemically aggressive environment.36
The similar functionality of spongin and chitin results from the analogical structural design of the fibers, which are secreted as a three dimensional network.
The fibers are built according to the principles of hierarchical arrangement thanks to which so called Bouligand structures can be formed.45 Bouligand as a first described this structural strategy in which nano- and microscale molecular chains form fibrils, which then are assembled in bundles, and afterwards are joint to form fibers. In case of chitin and spongin one can observe saccharide chains and α-chains of amino acids, respectively, forming fibrils of several nanometers in width. These are twisted and assembled in bundles, which afterwards are intertwisted again to form fibers several micrometers wide. The hierarchical arrangement of the fibers ensures high durability and elasticity, maintaining low bulk density at the same time.46 Chitin and spongin networks often play a similar role of a template, nucleator and cooperative modifier in biomineralization processes occurring intracellularly as well as extracellularly.47–49
Despite the analogical functional properties of chitin and spongin, those two materials differ in number of aspects (Figure 5).
Although chitin and spongin are similar at the angle of strategy of extracellular secretion (hierarchical character of Bouligand structure), chitin is rather involved in formation of exoskeletons, while spongin form internal endoskeletons and is of mesodermal origin (already discussed in Chapter 2.1).50 Moreover, chitin and spongin show different functional properties. As reviewed by Wysokowski et al.51 chitin is primarily found in organisms were mechanical resistance (hardness) is required, and is associated with inorganic phase (usually calcium carbonate or calcium phosphate),
e.g. dactyl club of the Mantis shrimp. On the other hand, this polysaccharide is a building material of e.g. squid pens, where both flexibility and hardness are necessary. As far as structure concerned, chitinous and sponginous fibers can be vividly compared to different types of Italian pasta (Figure 5). Chitin fibers found in sponges form tube-like structures with hollow interior reminding macaroni noodles, while spongin fibers are always full inside and can be compared rather to commonly known spaghetti.
Figure 5. Principal difference between structure of skeletal fibers in keratosan demosponges. Fibers of typical bath sponges (A) are made of spongin and possess no apertures within. Their morphology is similar to that of spaghetti (B). However, skeletons of
demosponges of Verongida order like Aplysina fistularis (C) are based on chitinous fibers (up to 100 µm in diameter), which are tube-like. Correspondingly, chitinous fibers of these sponges resemble the morphology of macaroni (D). Differences in chemistry and morphology
of skeletal fibers of keratosan sponges are the limiting factors for their practical use (scale bar: 1 cm) (adapted from 52).
Most importantly, spongin and chitin differ chemically. The chitinous skeleton is formed by amino-polysaccharide macromolecules, which can be often found together with silk-fibroin-like proteins and aspartic rich glycoproteins in molluscs.53 Spongin, on the other hand, is a unique protein found exclusively in Porifera phylum. Thanks to the genetic investigation of Exposito et al.54 and Aouacheria et al.55 a genetic evidence in NC1 domains were provided to prove that spongin is an evolutionary sister to the type IV collagen.
2.3 Chemistry of structural protein spongin
First experiments performed on demosponges are reported in work by French physician and chemist Geoffroy in 1705.56 In the simple experiment which involved burning of an ordinary representative of common bath sponges (most likely Hippospongia officinalis, H. communis or H. equine widely used at that time) he found a similarity of the odor of a burned sponge to a smell of burned hair or a horn, and as a conclusion pointed at the similarity of spongin to keratin.
Later, in 1843, Posselt57 conducted a similar experiment aiming at resolving of the chemical composition of the sponge skeleton. During heating up to 180-200 oC he found out that spongin is stable up to this temperature, but later it turns into ash containing silica, iron, phosphate of lime, gypsum, and small amount of potassium iodine. Posselt investigated also chemical stability using boiling water or various chemicals including, hydrochloric, nitric, and sulfuric acid and ammonia.57 He reported that spongin skeleton is undissolvable in non-concentrated acids and only prolonged bath in nitric acid, followed by a soaking in ammonia allowed to dissolve the sponge entirely.
Croockewit58 reported that except the presence of iodine, sulfur, and phosphorus sponginous skeleton of sponges is very similar to silk fibroin and sericin (protein created by silkworm). Moreover, as a first he proposed a formula describing spongin:
20(C39H62N12O17)+J2S3P10. Schlossberger,59 however, had some doubts to the statements reported by Posselt and Croockewit that spongin is chemically analogical to the fibroin. He supported his argument with the results of amino acid analysis of spongin and silk treated with diluted sulfuric acid, which in the former one yielded leucine and glycol, and in the latter one tyrosine and serine. What is more, spongin was not so easily diluted in Schweitzer’s reagent (ammoniacal solution of copper hydroxide) as silk fibroin. As conclusion Schlossberger suggested that difference may lay in the presence of sulfur, iodine, and phosphorus in the amino acid sequence comprising spongin. Similarly, Städeler also found difference in spongin and fibroin of silk through analysis of amino acids (from spongin glycine and leucine was isolated but no tyrosine as in the case of silk).60 He was also the first to propose name spongin for the horny protein of sponge.
Simultaneously, some experiments were focused on morphological rather than biochemical aspects of spongin of horny sponges. Gray61 in 1825 as first noticed the presence of siliceous spicules in sponges after he accidentally scratched the surface of glass by rubbing against it. The first who observed 3-dimensional network of fibers of keratose Australian sponges with use of optical microscope was Bowerbank.62 He reported on honeycombed network of minute fibers not exceeding 2.5 micrometers in diameter, and also described the cellular structure of the skeleton (Figure 6).
In 1900, Minchin63 published a “Treatise on Zoology” in which he observed distinct forms of spongin as a skeletal element: (i) cementum which works as a glue
joining the spicules into a complex network, and (ii) fibrillar spongin which he quite accurately compared to the elastic tissue of higher metazoans.
With development of analytical techniques it was also possible to investigate the amino acid composition of proteinaceous spongin. Clancey64 in 1926 conducted a comprehensive study on the composition of keratose sponge, pointing at errors in previous reports connected with both analytical methods and quality of the material which was variously macerated and bleached, eventually leading to partial decomposition of sponge specimens. Nevertheless, through his analysis he confirmed that spongin is rather analogous to collagens than to fibroin of silk. Block and Bolling65 performed a similar experiment involving analysis of amino acids yields but additionally compared the results with the composition of keratin originating from various sources such as gorgonin, turtle scutels, human skin and neurokeratin, which according to his result is the most similar to spongin.
Figure 6. Fragment of sponge, showing patches of fine reticulated structure of keratose sponge presented by Bowerbank (adapted from 62).
Spongin has to be also considered as a reservoir of halogen elements (particularly iodine and bromine) acquired and bonded from marine environment, and indeed it was numerously analyzed at this angle. The first experiment was conducted by Fyfe66 in 1819 who used starch as an indicator of the iodine presence. Bromine was found also in a commercially available sponges in a study performed by Hermbstädt.67 The research by Demselben68 indicated that the halogens exist mainly in form of potassium and magnesium salts, however Vogel69 stated the opposite – that iodine and bromine are bonded mostly as organic compound. Hundeshagen70 came out with similar observations and suggested that iodine is bonded to one of the amino acids forming iodotyrosine and called it jodospongin. He reported that sponges, as filter feeders, are able to collect elements like iodine from marine waters,
which can be found in sponge in amounts as high as 14% (identified in several species of Verongida order) and suggested that sponge plantations could become an alternative source of iodine. The confirmation for the organically bonded iodine can be found in observations by Wheeler et al.71 who in rather complicated procedure was able to extract 3,5-diiodtyrosine, or the so-called iodogorgoic acid from ordinary bath sponge. Dunnington72 not only confirmed the presence of iodine, bromine and chlorine in sponges (in his research the sponges were collected from Florida, Cuba and Bahama Islands) but also gave the exact percentage content equal to 0.603%, 1.307% and 1.060%, respectively.
Modern analytical methods and tools allow to further investigate the biochemical and structural nature of spongin. Spongin is secreted by epithelial cells called spongioblasts and as suggested by Gross et al.73, it can be formed in two distinct forms: spongin “A” that is fibrillar intercellular collagen of which purpose is to cement siliceous spicules together as well as play a role of an anchor which attach sponges to its substrata. It also shows similar axial periodicity of 625 Å which fits with characteristic range of axial period of collagens (600-650 Å); spongin “B” is the building material of the horny skeleton and is composed of large branched bundles of filaments of 10-50 µm in width (Figure 7).73 However, Bergquist26 in her comprehensive work on sponges disagreed with the division of spongin into two types. Instead, she stated that fibrillar collagen called spongin “A” is not peculiar to sponges and can be often found throughout the animal kingdom as collagen, therefore it should not be further used, in opposite to spongin “B”, which is unique to Porifera and can be used exclusively regarding sponges. Since spongin is a protein it is also important to consider the composition of amino acid residues in order to fully understand the nature of this biomaterial. Several authors analyzed spongin at the angle of amino acid content and it can be easily noticed that with time the accuracy of the measurement have been increasing, and more and more amino acids have been detected. In Table 3 the results of several experiments concerning the amino acid composition of spongin are presented. Although the authors used different marine sponge species all of them belonged to the Dictyoceratida order of Demospongiae. Additionally the table contains information about the composition of collagen extracted from ox bone, therefore a similarity of spongin to collagen can be observed in terms of specific amino acid content. Noteworthy, not all authors provide a specie name of tested sponges but simply refer to a “bath sponge”, which indicates that the examined skeleton most likely belonged to H. communis or Spongia officinalis.
Figure 7. (A) Spongin “A” and “B” observed in fresh sponge body of Spongia graminea by;74 (B) photomicrograph from polarizing microscope showing smear of fresh sponge;
(C) electron micrograph of spongin “A” revealing 600-700 Å axial period.
Table 3. The content of amino acids in spongin in various species of Dictyoceratida order (Demospongiae) according to various authors.
Percentage content according to various authors (per 100 g of spongin)
Amino acid
Bath sponge75
(1906)
H. communis64 (1926)
Bath sponge65
(1939)
S. graminea74 (1959)
H. communis41 (1974)
Ox-bone collagen76 (1955)
Alanine − 0.2 − 5.4 8.4 10.5
Arginine − 5.9 4.3 5.4 4.5 9.2
Aspartic acid 4.7 4.5 − 9.3 9.4 7.1
Cystine − trace 2.8 1.1 7 −
Diiodotyrosine − − trace − − −
Glutamic acid 18.1 18.4 − 8.9 7.9 −
Glycine 13.9 14.0 14.4 15.0 31.9 25.3
Histidine − 0.0 0.2 0.4 0.4 1.0
Hydroxylysine − − − 2.8 2.9 1.1
Hydroxyproline − − − 8.6 8.7 14.1
Isoleucine − − − 1.6 2.1 −
Leucine 7.5 − − 2.2 2.7 3.9
Lysine − − 3.0 2.5 3.4 4.1
Percentage content according to various authors (per 100 g of spongin)
Amino acid
Bath sponge75
(1906)
H. communis64 (1926)
Bath sponge65
(1939)
S. graminea74 (1959)
H. communis41 (1974)
Ox-bone collagen76 (1955)
Methionine − − − 0.3 Trace 0.8
Phenylalanine − − 3.3 1.2 1.0 2.9
Proline 6.3 5.7 − 5.8 6.7 14.7
Serine − − − 1.7 2.5 4.2
Threonine − − − 2.2 2.6 2.5
Tyrosine − 2.8 0.8 0.6 0.2 0.6
Valine − − − 1.9 3.0 2.7
One can notice that spongin is a member of collagen class due to similar amount of glycine equal to about 14% (except H. communis investigated by Garrone et al.42), hydroxyproline, tyrosine, arginine, lysine, histidine, threonine, and valine as compared with amino acid content of higher metazoans. High sugar content was also typically found in number of spongin containing sponges. In Spongia graminea, glucose, galactose, xylose, mannose, and arabinose were found in conjunction with spongin fibers. Junqua et al.41 found small amounts of galactosyl-hydroxylysine and much more substantial amounts of glucosylgalactosyl-hydroxylysine in three marine sponges (Ircinia variabilis, H. communis and Cacospongia scalarist).
Noteworthy, the measured content of some amino acids differ even within the same specie when measured by various authors. The difference might result from such factors as different advancement of available at the time analysis methods, different preparation methods of samples as well as various collection sites of sponge specimens.
When genetic studies became more accessible for the scientific community it was possible to investigate the parameters of spongin also at the level of DNA, and collect more reliable data. Exposito et al.54, through cDNA cloning compared NC1 domains (a characteristic sequence of amino acids responsible for chain bonding and their stabilization) of spongin of the fresh water sponge Ephydatia mülleri, (Haplosclerida) and type IV collagen and found a similarity between them. Even though sequence identity between these collagens is relatively small (except for short stretches), the NC1 region of both proteins can be divided into two subdomains (NC1-A and NC1-B) presenting about 27% identity. He also found similarity of spongin with nematode cuticular collagen through comparison of NC2 region, particularly in terms of cysteine residues, which were perfectly aligned. Remarkably, it was also suggested that spongin reflects two lines of evolution: one might have been exocollagens attaching sponges to the substratum (basal spongin described in Chapter 2.1), and the second might have been in internalization of such collagens, leading to the differentiation of basement membrane collagens. The statement was
supported with the fact that almost all sponges (except Homoscleromorpha class) are lacking basement membranes, and similarities between spongin, nematode cuticular, and basement membrane type IV collagens. Additionally, vertebrate FACIT (Fibril Associated Collagens with Interrupted Triple helices) and FACIT- related collagens seem to be evolutionarily related to nematode cuticular collagens.
All consist of several short collagenous domains, with similar C-terminal noncollagenous (NC1) domains as well as conserved cysteine residues at the COL1–
NC1 junctions.54
Similar results, by comparing the NC1 regions of DNA chain of E. mülleri spongin and collagen type IV were obtained by Aouacheria et al.55 who compared modular structure, examined primary sequence features, and modeled structure of the NC1 domain. Interestingly, in Homoscleromorpha sponges which can be characterized with well-developed epithelium, a basement membrane type IV collagen is present (as in the case of all Eumetazoa), and it was stated that perhaps this group of sponges represent first stage of tissue differentiation during animal evolution.
Amazingly, Exposito et al. in 2008 by studying the publicly available genome data of the choanoflagellate Monosiga brevicollis, the demosponge Amphimedon queenslandica, the cnidarians Hydra magnipapillata (Hydra) and Nematostella vectensis (sea anemone) presented an evidence for evolutional continuity of characteristic modular structure of B clade collagens spanning from sponges to humans.77
Considering the above, it is easy to notice that spongin shares with collagen number of biochemical features, particularly the amino acid composition. It can be hypothesized that spongin is also similarly build in terms of amino acids sequence, which in case of collagen is constructed according to a Gly(glycine)-X-Y triplet motif, where X or Y position is usually occupied by hydroxyproline (Hyp) and the remaining position by any of the present amino acids.78 On the other hand, the remarkable thermal and chemical stability of spongin suggests that the poriferan protein shares some characteristics of keratin as well. Most likely, the enhanced thermal performance results from the stabilizing effect of cross links formed between cysteine residues and hierarchical strategy according to which the fibrils are aligned (described in details in chapter 2.6). Based on the above considerations spongin can be considered as an intermediate (hybrid) biomaterial between collagen- and keratin- like proteins, for which a hypothetical chemical model of fibers was proposed in Figure 8.
Figure 8. Structure of spongin fiber and possible amino acid composition of spongin α-chains forming triple helix, strengthened via sulfur bridges.
2.4 Structural properties of spongin–based skeletons
Sponges are filter feeders, meaning that they have to pump massive amounts of water through their bodies and filter out nutrients even at the sea floor where food availability is rather poor. In order to perform the pumping in the most optimal way, the skeleton of sponges, either partly or fully mineralized, was evolutionary shaped and takes advanced structural designs. Regardless the material the skeleton is built of (chitin like in Verongida order,36,79–81 or spongin as in Dictyoceratida and Dendroceratida order), it is shaped in a way that ensures light and porous construction for filtering, but at the same time is mechanically resistant and durable to withstand water currents.
The remarkable functionality of the horny skeleton of Demosponges is owed to two structural designs comprising the spongin fibers from nano- to macrolevel. At nanoscale a hierarchical arrangement of the fibers was identified, which can be described as a group of molecular units/aggregates that are in contact with other phases, which in turn are similarly assembled at increasing length scales.82 In the case of collagenous fibers of spongin, α-chains of amino acids form fibrils, which then form bundles of fibrils which are assembled to form fibers of about 20 µm in diameter (Bouligand structure). Generally, the number of hierarchy levels comprising any fiber strongly influences stiffness, strength and toughness of the material (the greater the number of levels in the hierarchy the less stiff and more strong the material is), what was explained by, so called, a Russian doll model implemented by Ji and Gao (Figure 9).83,84
The spongin fibers constructed in hierarchical manner at macroscale are organized in 3-dimensional anastomosed network following the design of cellular arrangement. This foam-reminding structural solution allows the material to have good mechanical properties at low weight.85 According to the definition given by Chen et al.82 a cellular solid is an interconnected network of struts or plates that form the faces of cells. Cellular solids are characterized by the shape and distribution of the cells and can be classified as open or closed cell forms. Horny skeleton of demosponges is an example of the open cell form (in contrary to cork, which displays closed cell form).
Figure 9. Schematically presented hierarchical arrangement of marine sponge fibrous skeleton of H. communis from α-chains of amino acids to fibers
according to a Russian doll model (adapted from 86).
The behavior of cellular solid during crushing was illustrated in Figure 10, in which 3 typical regions can be observed: (i) an elastic region accompanied with linear elastic strut bending, (ii) a collapse plateau related to an elastic buckling of the open-cell, and (iii) a densification region during which cell walls/struts crush together.
To understand the deformation and failure mechanism of sponge skeleton during compression it can be compared to the behavior of cells in three-dimensional cellular solids (foams) what was explained by Gibson.87 Although a cubic cell was used to illustrate the mechanisms analogical result can be obtained for any cell geometry.
Figure 10. A typical stress and strain curve a cellular materials supported with images of cell behavior.87
The relationship between the relative stiffness (E*/Es) and relative density (ρ*/ρs) for an open cell foam can be given as:
E*
Es
=C
1 ρ*ρs 2
(1)
Where E* is a Young’s modulus, Es is the stiffness of the fully dense solid, ρ* is a measured density and ρs denotes a density of the solid making up the walls or struts (in case of marine sponge skeleton spongin fibers). The analysis gives the dependence of the Young’s modulus on the solid modulus and the relative density.
The discussed parameters can be used in construction of materials property chart where Young’s modulus is plotted versus density of various materials (Figure 11).
Using available data on mechanical parameters of spongin-based skeleton it was possible to add a spongin-based skeleton related area in this plot for the first time.
Figure 11. Plot of the Young’s modulus versus density for various biomaterials including spongin-based skeletons (adapted from 88).
Sponge skeletons included into the group of natural cellular materials are located in the lower left quarter of the chart, indicating both low density and low Young’s modulus. That means that the skeletons of sponges belong to the one of the most light and porous natural materials, which gives them their softness and explains their attractiveness as a bathing product.
In natural conditions the mechanical performance of the skeletons of several species of sponges are enhanced by spicules. The siliceous spicules found in most sponges fulfil important reinforcing role of the connective tissue. According to the early research performed by Koehl,89 siliceous spicules stiffen connective tissues in a manner analogous to the stiffening of pliable polymers by filler particles. The study identifies several parameters of the spicules (volume fraction, shape, size and orientation) on the tensile stiffness of spiculated tissues and following relations were found: (i) spicules increase the stiffness of pliable connective tissues, probably by mechanisms analogous to those by which space-occupying filler particles stiffen deformable polymers; (ii) the greater the volume fraction of spicules, the stiffer the tissue; (iii) the greater the surface area of spicules per volume of tissue, the stiffer the tissue; (iv) spicules that are anisometric in shape have a greater stiffening effect
parallel to their long axes; (v) spicules with very high aspect ratios appear to act like reinforcing fibers; (vi) spicule-reinforced tissues exhibit stress-softening behavior75.
The mechanical parameters of demosponge skeleton are crucial from the applicability point of view because establishing of factors for proper selection of sponges during aquaculture and later harvesting directly influences preparation of high-quality bath sponges.90 Moreover, systematization of mechanical parameters allow to compare and to select the best specimens suited to a given application.
Louden et al.91 conducted a detailed study on several species of bath sponges Rhopaloeides odorabile, Coscinoderma sp. and three commercial species, H. lachne, Spongia 1, and Spongia 2. The study involves analysis of several mechanical parameters including: (i) firmness (the stress required to compress the sponge by 25%) which has direct implications for the feel of sponge; (ii) compression modulus (stress required to compress the sponge by 65% divided by its firmness) which is a measure of the sponge's ability to cushion or support a weight; (iii) elastic limit (the point at which damage first occurs) considered a more valuable and appropriate measure of sponge strength; (iv) modulus of elasticity which is the rigidity or stiffness of a material; (v) modulus of resilience which was estimated as the energy absorbed by a material when stretched to its elastic limit and the energy released when returning to its original form; (vi) absorbency and water absorption efficiency.
Significant differences were found between examined samples and a unique profile was determined for each of them. R. odorabile was the firmest (37.8±4.3 kPa), strongest (157.4±17.3 kPa), and most rigid (838.7±53.5 kPa) of tested species, while Coscinoderma sp. was one of the softest sponges (7.3±1.1 kPa) and had the highest elastic energy (30.5±3.5 kJ/m3) and water retention efficiency (40.1±1.4%) of all.
Among the commercial species, H. lachne was the softest (3.2±0.3 kPa), weakest (36.3±3.1 kPa), and most absorbent sponge (31.0±1.1), while Spongia 1 and Spongia 2 had intermediate quality characteristics for all measured parameters.
The measured parameters provide information regarding field of use of a given sponge specimen. The possible applications of these animals are discussed in a following subchapter.
2.5 Spongin as biomaterial – from historical applications to tissue engineering The attractive features of marine sponges characteristic for species which are today recognized as bath sponges (H. communis, S. officinalis etc.) where recognized already in ancient times. They were collected by ancient Greeks and first annotation on use of sponges was already found in works of Homer in 8th century BC. In 4th and 5th century BC their medical applications were widely described by Hippocrates, while Aristotle focused on the zoological aspects of sponges, which at the time were considered to possess features of both animal and plant kingdom. Sponges were also
acknowledged in the King James Version of the Bible in the accounts of Christ crucifixion in Mt 27:48; Mk 15:36; Jn 19:29.92
An accounts of sponge knowledge in Greek antiquity were investigated comprehensively and in details by Voultsadou, who analyzed texts written in classical Greek from the 8th to the end of the 1st century BC using search engine of digital library of University of California.93 Through his findings it was possible to learn, that Greeks collected large and accurate knowledge concerning such aspects of sponges as diversity, morphology, symbiotic relationships, characteristic features, habitat and distribution, but most importantly, they found an impressive amount of application of sponges particularly in a field of medicine and pharmacology (Table 5).
Table 5. Various applications of sponges in ancient cultures.79
Area of application Description
Cleansing Wet sponges were used for washing body, also in healing baths, in the same manner they are used nowadays.
Medicine and pharmacology
Sponges soaked in cold water were placed on the heart of somebody who had fainted.
In case of various head diseases or pains in this part of the body, sponges were soaked in hot water and apply to ease the pain.
Before exposure to sun, large and wet sponges were placed on head to prevent heat-stroke.
When soaked in honey they were put inside ear for treatment of otorrhoea (ear inflammation).
A special devices were constructed using twisted and wounded with linen thread sponge to remove polypus from nostrils.
During clyster therapy sponges were plugged inside anus in order to keep a proper liquid in the colon until finished.
Before applying a medicine, wounds and sores were cleaned with sponges in order to remove the secreted body fluids. When soaked in oil, the skeletons of horny sponges were applied to sear sores or scars.
Connection of antibacterial effect of honey was again used with use of sponges in order to treat hemorrhoids.
The collected sponges were often used in treatment of various gynecological diseases and pains of uterus. In such cases the animal was soaked in oil or hot water and placed at aching site of the body. In case of gynecological diseases sponges were also used as pharmaceutical ingredient in two ways: first, when mixed with a seaweed and seal rennet were applied to fumigate genitals; in second situation, burned sponge was mixed with wine and recommended for drink to treat intense or prolonged blood flow during women’s menstruation. It can be suspected that ancient Greeks unconsciously noticed the antibacterial effects of iodine present in marine organisms like weed or sponges. Similar observations were made by ancient Chinese who used burned sponge as source of iodine in treatment of enlarged goiter.
Various uses Sponges were found among common household items and were used in number of everyday situations. Besides that, Greeks used them in military applications, for better comfort fitting of a warrior’s armor, or to prevent fire on wooden war machines.
In Egypt, gold was mined and purified in a complicated process and sponges were used to collect minute pieces of the metal.
Through the following ages the use of sponges did not change significantly, until the revolution in medicine and surgery at the break of XVIII and XIX century. At that time, sponges were appreciated due to their softness, high compressive strength, ability to retain shape, and high sorption rates. For these reasons they were used as a compression bandage for pressing open sinuses, in overcoming strictures of body passages (e.g. rectum), dilating of cervix uteri,94–98 and in form of sponge tents applied in uterus in order to expand the cavity and examine the issues.99,100 Some report prove that fragments of sponge skeleton were used as a small prostheses in early “plastic surgery” (Figure 12B).101 Moreover, when physicians became aware of the impact of bacteria and necessity of sterilization, they applied various methods for preparation of aseptic sponge,102–104 which otherwise emitted unpleasant odor and were a cause of inflammation.105,106
Finally, biocompatibility of fibrous spongin was noticed and used in treatment of wounds. First reports refer to an ability of sponges to support the young vessels and produce gradual pressure during absorption of fluids and swelling.94,98,107 The initial
studies involving application of sponginous skeleton of sponges in treatment of damaged soft tissue refer to so called sponge-grafting. Hamilton108 was the first to report on use of thin slices of sponges as support for granulating tissue until it was completely covered with epidermis. The idea was supported with unique features of spongin skeleton including high porosity imitating interstices of the fibrous network in a blood or fibrous-clot, ability to be resorbed by the patient’s body and possibility to be adjusted to any surface.107 Hamilton reported four cases successfully treated via sponge-grafting. In one of them, a woman was subjected to a surgery for removal of a mamma tumor during which large area of skin was removed. The deleted skin was replaced with thin slice of an aseptic sponge skeleton, which after ten days from a surgery was noticed to be vascular, and 3 months later was covered with epithelial tissue (Figure 12A).
The success of Hamilton, encouraged other physicians to experiment with sponge- grafting in number of other, often severe cases.109–114 However, difficulties in maintaining spongin skeletons septic, slow or completely ineffective recovery of damaged tissue as well as increasing quality of general standards of public health service, ceased the utilization of spongin as a porous support in wound healing applications what can be observed in diminished number of published papers.115 Sponges remained simple bathing product recognized for their softness, durability and cleansing properties.
However, only recently it was suggested that spongin and chitin, which work as a scaffolds for biomineralization of inorganic materials in sponges (silica or calcium carbonate)34,35,50,116 share a common evolutionary purpose with collagen, namely as an unified template for biomineralization of siliceous and calcareous phosphate skeletons (bones).117
If spongin possesses similar characteristics to collagen then perhaps it is possible to use the skeleton of horny sponges as its alternative in a medical field of tissue engineering? That could be a question that Green118 asked himself when as a first examined the behavior of human bone-building cells in the presence of fibrous spongin skeleton. He investigated attachment, growth and differentiation of human osteoprogenitor cells onto the sponginous template isolated from S. officinalis, and reported that the material can be characterized with number of advantageous for tissue engineering features, including: (i) the ability to hydrate to a high degree, which is favorable in respect to cell adhesion; (ii) the presence of open interconnected channels created by the fibers network; (iii) the collagenous character of the fibers; (iv) the tremendous diversity of skeletal architectures which are strikingly similar to internal parts of human bone and are almost impossible to replicate via available synthesis methods; and (v) provides structural environment conducive for proliferation, as demonstrated by bridging of the interfibrilar spaces.119 Moreover, in contrary to commonly used sources of collagen (usually of bovine origin), spongin isolated from marine sponges does not bear the risk of diseases
