María S. Fernández

Polysaccharides and proteoglycans in calcium carbonate-based biomineralization.

Biomineralization is a widespread phenomenon in nature leading to the formation of a variety of solid inorganic structures by living organisms, such as intracellular crystals in prokaryotes, exoskeletons in protozoa, algae, and invertebrates, spicules and lenses, bone, teeth, statoliths, and otoliths, eggshells, plant mineral structures, and also pathological biominerals such as gall stones, kidney stones, and oyster pearls. These biologically produced biominerals are inorganic-organic hybrid composites formed by self-assembled bottom up processes under mild conditions, showing interesting properties, controlled hierarchical structures, and remodeling or repair mechanisms which still remain to be developed into a practical engineering process. Therefore, the formation of biominerals provides a unique guide for the design of materials, especially those that need to be fabricated at ambient temperatures. In biominerals, the small amount of organic component not only reinforces the mechanical properties of the resulting composite but also exerts a crucial control on the mineralization process, contributing to the determination of the size, crystal morphology, specific crystallographic orientation, and superb properties of the particles formed. Therefore, biological routes of structuring biominerals are becoming valuable approaches for novel materials synthesis. Although several principles are applicable to the majority of the biominerals, herein we will focus on the role of polysaccharide polymers in calcium carbonate-based biominerals. As a general principle, the assembly of these biominerals consists of a four-stage process. It starts with the fabrication of a hydrophobic solid organic substrate or scaffolding onto which nucleation of the crystalline phase takes place closely associated with some polyanionic macromolecules. Crystal growth is then controlled by the addition of gel-structuring polyanionic macromolecules, and finally mineralization arrest is accompanied by the secretion of a new inert scaffolding of the same type or the deposition of other hydrophobic inhibitory macromolecules. Currently, a large number of proteins have been described which are involved in the control of biomineralization. These proteins are usually highly negatively charged and contain carboxylate, sulfate, or phosphate as functional groups, which may bind Ca ions and could control crystal nucleation and growth by lowering the interfacial energy between the crystal and the macromolecular substrate. However, the precise mechanism involved in controlling crystal nucleation, growth, and morphology is far from being understood. Combinatorial biology techniques have been recently developed for testing the ability of randomly generated peptides to bind different substrates or ions, thus allowing a correlation between peptide structure and ion binding affinity. However, the main focus is on the role of the backbone structure of the polymer due to the primary structure of the protein, because the synthetic technology does not allow the formation of post-translational modifications, such as sulfation and phosphorylation, which do occur in the eukaryotic cell. Even so, the occurrence of negatively charged groups in macromolecules involved in biomineralization, mainly derived from acidic amino acids, has inspired many researchers to produce synthetic polymers having such groups in order to control the size, orientation, phase, and morphology of inorganic crystals. However, since Abolins-Krogis’ work, a slow but increasing interest has been developed to explore the role of polysaccharides in biomineralization, despite the fact that their involvement in biomineralization seems to appear very early in evolution. There is no single type of polysaccharide associated with biominerals, but such polysaccharides are mainly hydroxylated, carboxylated, or sulfated or contain a mixture of these functional moieties.

Collagens of the Chicken Eggshell Membranes

An immunohistochemical analysis of the eggshell membranes shows the occurrence of type X collagen while type I collagen was not detected by using an appropriate monoclonal antibody with untreated shell membranes. A positive immuno-reaction for type I collagen was obtained after digestion of the shell membranes with pepsin. These observations indicate the possibility that type I collagen epitope was masked by type X collagen and that type X collagen may serve as an inhibitory boundary for biomineralization.

Secretion pattern, ultrastructural localization and function of extracellular matrix molecules involved in eggshell formation.

The chicken eggshell is a composite bioceramic containing organic and inorganic phases. The organic phase contains, among other constituents, type X collagen and proteoglycans (mammillan, a keratan sulfate proteoglycan, and ovoglycan, a dermatan sulfate proteoglycan), whose localization depends on a topographically defined and temporally regulated deposition. Although the distribution of these macromolecules in the eggshell has been well established, little is known about their precise localization within eggshell substructures and oviduct cells or their pattern of production and function during eggshell formation. By using immunofluorescent and immuno-ultrastructural analyses, we examined the distribution of these macromolecules in oviduct cells at different post-oviposition times. To understand the role of proteoglycan sulfation on eggshell formation, we studied the effects of inhibition of proteoglycan sulfation by treatment with sodium chlorate. We showed that these macromolecules are produced by particular oviduct cell populations and at precise post-oviposition times. Based on the precise ultrastructural localization of these macromolecules in eggshell substructures, the timing of the secretion of these macromolecules by oviduct cells and the effects on eggshell formation caused by the inhibition of proteoglycan sulfation, the putative role of mammillan is in the nucleation of the first calcite crystals, while that of ovoglycan is to regulate the growth and orientation of the later forming crystals of the chicken eggshell.

Eggshells are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules

The avian eggshell is a composite bioceramic which is formed by a controlled interaction of an organic and an inorganic phase. The organic phase contains, among other constituents, type X collagen and proteoglycans, mainly keratan and dermatan sulfate. Understanding the principles governing the synthesis and temporo-spatial distribution of such macromolecules, and their influence on the organization of the crystalline phase, is an essential aspect of establishing the biological basis of the quality of eggshell, both as an embryonic chamber and as a natural food package. In the present study, we have examined the process of eggshell formation by immunohistochemistry, scanning electron microscopy and energy dispersive X-ray microanalysis. Precise sites and timing of secretion were established for the deposition of particular macromolecules. Type X collagen is detected at the very first moment of shell membrane formation. The appearance of keratan sulfate coincides with the appearance of mammillae, while dermatan sulfate is deposited later, coincident with shell matrix deposition. We propose that keratan sulfate, due to its precise localization, temporal appearance and calcium-binding affinity, relates to the maintenance of calcium reserve bodies, the primary source of calcium for the embryo. On the other hand, dermatan sulfate may control crystal growth, resulting in a preferential orientation of calcite crystals within the palisade layer.

Role of type X collagen on experimental mineralization of eggshell membranes.

Type X collagen is a transient and developmentally regulated collagen that has been postulated to be involved in controlling the later stages of endochondral bone formation. However, the role of this collagen in these events is not yet known. In order to understand the function of type X collagen, if any, in the process of biomineralization, the properties of type X collagen in eggshell membranes were further investigated. Specifically, calvaria-derived osteogenic cells were tested for their ability to mineralize eggshell membranes in vitro. Immunohistochemistry with specific monoclonal antibodies was used to correlate the presence or absence of type X collagen or its propeptide domains with the ability of shell membranes to be mineralized. The extent of mineralization was assessed by Von Kossa staining, scanning electron microscopy and energy-dispersive spectroscopy. The results indicate that the non-helical domains of type X collagen must be removed to facilitate the cell-mediated mineralization of eggshell membranes. In this tissue, intact type X collagen does not appear to stimulate or support cell-mediated mineralization. We postulate that the non-helical domains of type X collagen function in vivo to inhibit mineralization and thereby establish boundaries which are protected from mineral deposition.

Sulfated polymers in biological mineralization: a plausible source for bio-inspired engineering

Biomineralization leads to the formation of inorganic crystals with unique, ordered, refined shapes that are regulated by specific macromolecules. This process has been a source of inspiration for exploring novel approaches to the fabrication of inorganic-based surfaces and interfaces. Among those macromolecules, sulfated polymers, referred to as proteoglycans, have not received enough attention, although there is increasing evidence of their widespread occurrence in biominerals. Here we examine the available information on the nature, distribution and possible role of sulfated polymers in biomineralization, and highlight new directions to stimulate further research activities.

The Avian Eggshell Extracellular Matrix as a Model for Biomineralization

The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest that the dermatan sulfate proteoglycans may be important in modulating crystal morphology in the hen eggshell and correlate with mineralization-modulating biomolecules from other calcified tissue, which are generally anionic.

Effect of Sulfate Content of Biomacromolecules on the Crystallization of Calcium Carbonate

Natural composite bioceramics such as bone, teeth, carapaces and shells contain organic and inorganic moieties, with the organic matrix components directly involved in the precise formation of these structures. We have previously shown that chicken eggshell contains two main sulfated polymers (proteoglycans), referred to as mammillan and ovoglycan which are involved in nucleation and growth of the eggshell calcite crystals. They differ on their anionic properties due to the carboxylate and sulfate content of their glycosaminoglycan component. Based on biological and biochemical evidences, the putative role of mammillan, a keratan sulfate proteoglycan, is in the nucleation of the first calcite crystals, while that of ovoglycan, a dermatan sulfate proteoglycan, is to regulate the growth and orientation of the later forming crystals of the chicken eggshell. In this communication, a systematic study of the influence of variable concentrations of glycosaminoglycans differing in their sulfation status on the morphology, size and number of calcium carbonate crystals after crystallization on microbridges from a calcium chloride solution under an atmosphere of ammonium carbonate at different pH is presented. Depending on the pH and concentration, the variation of sulfation status drastically changed the morphology, size and number of calcite crystals. The produced calcite particles with various morphologies are promising candidates for some novel materials with desirable shape- and texture-depending properties.

Eggshell membrane as a biodegradable bone regeneration inhibitor

The efficiency of chicken eggshell membranes combined with a minimally invasive small osteotomy procedure of the ulna to accomplish an efficient release of the radius so that it can continue to grow in an unstressed manner was tested in rabbits. Eggshell membranes were extracted from chicken eggs, rinsed, dried and sterilized with ethylene oxide for 24 h. For reactivity testing, four separate subcutaneous pockets were created in 10 rats in the paravertebral region by blunt dissection and eggshell membranes were implanted in two of them. After 1–16 weeks, the implants were retrieved with the surrounding soft tissues and submitted to histological examination. Subsequently, 10 rabbits were anaesthetized and a complete 0.5 mm wide osteotomy was performed in both the right and the left distal ulna. A piece of eggshell membranes was interposed in the osteotomy site of one ulna. The opposite osteotomized ulna was left as a negative control. The rabbits were injected with oxytetracycline at the time of surgery and this was repeated every 7 days for labelling new bone formation. After 1–16 weeks, ulnar osteotomized regions were histologically examined. After histological, fluorescence microcopy and radiological evaluation, we demonstrate here for the first time that eggshell membranes as interpositional material in rabbit osteotomized ulnar experiments acted as an active barrier against bone bridging. The degradation of the eggshell membrane, due to host reaction, appeared sufficiently late to cause the desirable delay of bone healing that is compatible with the time needed for a corrective response. Copyright

Proteoglycan Occurrence in Gastrolith Of The Crayfish Cherax quadricarinatus (Malacostraca: Decapoda)

ABSTRACT Biomineralized structures are hybrid composites formed and stabilized by the close interaction of the organic and the inorganic phases. Crayfish are good models for studying biomineralization because they develop, in a molting-mineralization cycle, semi-spherical mineralized structures referred to as gastroliths. The organic matrix of these structures consists of proteins, polysaccharides, and lipids. Chitin is the main polysaccharide and is concentrically arranged as fibrous chitin-protein lamellar structures. Although several proteins and low-molecular weight phosphorylated components have been reported to be involved in gastrolith mineralization, the occurrence and role of proteoglycans have not been fully documented. We have immunologically analyzed the proteoglycans in gastrolith matrix extracts and histological cross-sections of the gastrolith, and the forming epithelium during premolt and postmolt stages. The results indicate that gastroliths contain proteoglycans that have dermatan-, chondroitin-4- and 6-, and keratan sulfate glycosaminoglycans. These macromolecules are closely associated with the mineral phase of the gastrolith and are easily removed by decalcification procedures. There is also evidence to indicate that epithelial secretion of some of these molecules is temporally regulated during the molting cycle. However, the precise role of these macromolecules in the calcification and stabilization of the amorphous calcium carbonate phase of the gastrolith remains to be established.