Chemical Investigation of the Calcareous Marine Sponge Pericharax heteroraphis, Clathridine-A Related Derivatives Isolation, Synthesis and Osteogenic Activity.

As a result of screening a panel of marine organisms to identify lead molecules for the stimulation of endochondral bone formation, the calcareous sponge Pericharax heteroraphis was identified to exhibit significant activity during endochondral differentiation. On further molecular networking analysis, dereplication and chemical fractionation yielded the known clathridine A-related metabolites 3-6 and the homodimeric complex (clathridine A)2 Zn2+ (9), together with the new unstable heterodimeric complex (clathridine A-clathridimine)Zn2+ (10). With the presence of the zinc complexes annotated through the LC-MS analysis of the crude extract changing due to the instability of some metabolites and complexes constituting the mixture, we combined the isolation of the predicted molecules with their synthesis in order to confirm their structure and to understand their reactivity. Interestingly, we also found a large quantity of the contaminant benzotriazoles BTZ (7) and its semi-dimer (BTZ)2CH2 (8), which are known to form complexes with transition metals and are used for preventing corrosion in water. All isolated 2-aminoimidazole derivatives and complexes were synthesized not only for structural confirmation and chemical understanding but to further study their bioactivity during endochondral differentiation, particularly the positively screened imidazolone derivatives. Compounds leucettamine B, clathridine A and clathridimine were found to increase type X collagen transcription and stimulate endochondral ossification in the ATDC5 micromass model.

Organic Matrices of Calcium Carbonate Biominerals Improve Osteoblastic Mineralization.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO3 biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

Dietary supplementation with nacre reduces cortical bone loss in aged female mice.

Aging is associated with detrimental bone loss leading to fragility fractures in both men and women. Notably, a majority of bone loss with aging is cortical, as well as a large number of fractures are non-vertebral and at the non-hip sites. Nacre is a product of mollusks composed of calcium carbonate embedded in organic components. As our previous study demonstrated the protective effect of nacre supplementation on trabecular bone loss in ovariectomized rats, we sought to evaluate the effect of dietary nacre on bone loss related to aging in female mice which do not suffer true menopause as observed in women. The current study compared the effect of a 90-day long nacre-supplemented diet to that of Standard or CaCO3 diets on both bone mass and strength in 16-month-old C57BL/6 female mice. Multiple approaches were performed to assess the microarchitecture and mechanical properties of long bones, analyze trabecular histomorphometry, and measure bone cell-related gene expressions, and bone turnover markers. In the cortex, dietary nacre improved cortical bone strength in line with lower expression levels of genes reflecting osteoclasts activity compared to Standard or CaCO3 diets (p < 0.05). In the trabeculae, nacre-fed mice were characterized by a bone remodeling process more active than the other groups as shown by greater histomorphometric parameters and osteoblast-related gene expressions (p < 0.05). But these differences were not exhibited at the level of the trabecular microarchitecture at this age. Collectively, these data suggest that dietary nacre should be a potential candidate for reducing aging-associated cortical bone loss in the elderly.

Preclinical safety study of nacre powder in an intraosseous sheep model.

Objectives: The purpose of this preclinical study was to evaluate the safety, the local tissue effects and bone healing performance (osteoconduction, osseointegration) of nacre powder in a sheep intraosseous implantation model. This represents the first preclinical study to assess nacre safety and efficacy in supporting new bone formation in accordance with the ISO 10993 standard for biomedical devices. Methods: The local tissue effects and the material performance were evaluated 8 weeks after implantation by qualitative macroscopic observation and qualitative as well as semiquantitative microscopic analyses of the bone sites. Histopathological characterisations were run to assess local tissue effects. In addition, microarchitectural, histomorphometric and histological characterisations were used to evaluate the effects of the implanted material. Results: Nacre powder was shown to cause a moderate inflammatory response in the site where it was implanted compared with the sites left empty. The biomaterial implanted within the generated defects was almost entirely degraded over the investigated time span and resulted in the formation of new bone with a seamless connection with the surrounding tissue. On the contrary, in the empty defects, the formation of a thick compact band of sclerotic bone was observed by both microarchitectural and histological characterisation. Conclusions: Nacre powder was confirmed to be a safe biomaterial for bone regeneration applications in vivo, while supporting bone formation.

Isolation, Synthesis and Absolute Configuration of the Pericharaxins A and B, Epimeric Hydroxy-Polyene Glycerol Ethers from the Calcarean Sponge Pericharax heteroraphis.

Naturally occurring epimeric hydroxy-polyene glycerol ether pericharaxins A (1a) and B (1b) were isolated from the calcarean sponge Pericharax heteroraphis. The structural and stereochemical characterization of both diastereoisomers were established on the basis of spectroscopic data analysis and total synthesis in seven steps. The mixture of pericharaxins A (1a) and B (1b) was proven to be epimeric by chiral-phase HPLC analysis of both synthetic and natural samples. Further separation of the epimers and application of Mosher's method to the synthetic compounds allowed unequivocal absolute configuration assignment. While natural products and the synthetic intermediates were shown to be non-cytotoxic on the HCT116 cell line, the endochondral differentiation activity using human type X collagen transcription activity in ATDC5 cells is interesting.

Organic Matrix and Secondary Metabolites in Nacre.

Nacre, also called mother-of-pearl, is a naturally occurring biomineral, largely studied by chemists, structural biologists, and physicists to understand its outstanding and diverse properties. Nacre is constituted of aragonite nanograins surrounded by organic matrix, and it has been established that the organic matrix is responsible for initiating and guiding the biomineralization process. The first challenge to study the organic matrix of nacre lays in its separation from the biomineral. Several extraction methods have been developed so far. They are categorized as either strong (e.g., decalcification) or soft (e.g., water, ethanol) and they allow specific extractions of targeted compounds. The structure of the nacreous organic matrix is complex, and it provides interesting clues to describe the mineralization process. Proteins, sugars, lipids, peptides, and other molecules have been identified and their role in mineralization investigated. Moreover, the organic matrix of nacre has shown interesting properties for human health. Several studies are investigating its activity on bone mineralization and its properties for skin care. In this review, we focus on the organic constituents, as lipids, sugars, and small metabolites which are less studied since present in small quantities.

Protective Effect on Bone of Nacre Supplementation in Ovariectomized Rats.

Nacre has emerged as a beneficial natural product for bone cells and tissues, but its effect was only studied by gavage in the ovariectomized mouse model. We sought to assess the antiosteoporotic effect of nacre through a nutritional supplementation in the ovariectomized rat model. Sixteen-week-old female Wistar rats were either Sham-operated or bilateral ovariectomized (OVX) and then fed with standard diet (Sham and OVX groups) or standard diet supplemented with either 0.25% CaCO3 or nacre (OVX CaCO3 and OVX Nacre group, respectively) for 28 days (n = 10/group). The bone microarchitecture was assessed at appendicular and axial bones by micro-computed tomography (µCT). Histomorphometric analysis was performed to determine cellular and dynamic bone parameters. Bone metabolism was also evaluated by biochemical markers and gene expression levels. Nacre-based diet prevented the OVX-induced bone loss better than that of the CaCO3 supplement, given the significant changes in trabecular bone volume fraction (BV/TV) both at the femoral distal metaphysis (difference, 35%; p = 0.004) and at the second lumbar spine (difference, 11%; p = 0.01). Trabecular osteoclast surfaces (Oc.S/BS) were also 1.5-fold lower at the tibial proximal metaphysis in OVX Nacre group compared with OVX CaCO3 group (p = 0.02). By principal component analysis (PCA), OVX Nacre group formed a cluster away from OVX group and with a trend closest to Sham group. These data were consistent with biological measurements demonstrating a positive profile related to nacre supplementation, which blunted an increase in serum CTX level and enhanced serum P1NP secretion 14 days post-OVX compared with CaCO3 supplementation. Bmp2 mRNA expression in OVX Nacre group was +1.76-fold (p = 0.004) and +1.30-fold (p = 0.20) compared with OVX and OVX CaCO3 groups, respectively. We conclude that supplementation with nacre could effectively limit bone loss induced by estrogen deficiency just after OVX in rats by modulating the negative imbalance of bone turnover. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Induction of Osteogenic MC3T3-E1 Cell Differentiation by Nacre and Flesh Lipids of Tunisian Pinctada radiata.

The flesh of the Pinctada radiata pearl oyster from coastal Tunisia is considered as a high source of n-3 and n-6 and its shell nacre layer is a promising osteogenic biomaterial. Fatty acid (FA) analysis showed that the major components found in total FA (TFA) were 14:0, 16:0, and 18:0 saturated FA (SFA); 16:1, 18:1, and 20:1 monoenoic FA; 20:4n-6 (ARA), 22:5n-3 (DPA). Characteristically high levels of 20:5n-3 (EPA) and 22:6n-3 (DHA) (6.53-89.75 mg/100 g TFA) polyunsaturated FA (PUFA) were found, respectively, in the TFA of nacre and flesh. Evaluated the effects in vitro of lipids extracted from nacre (Ln) and from flesh (Lc) of P. radiata on growth and the differentiation of osteoblasts. Cytotoxicity tests (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide [MTT] and lactic acid dehydrogenase c [LDH]) demonstrated that both extracts are nontoxic. Alizarin Red staining was used in an osteoblast differentiation model using the osteoblast MC3T3-E1 cell line. It showed that the FA of both extracts induced osteoblast differentiation leading to mineralization. Reverse transcription-polymerase chain reaction (RT-PCR) showed a significantly higher expression of osteocalcin (Bglap) and runt-related transcription (Runx2) in MC3T3-E1 cells in the presence of Ln. No difference of osteopontin (Spp1) and Collagen type I (Col1a1) genes compared to the control was observed. In conclusion, these results supported, obtained from our in vitro experimental model used, the interest/potential of lipids extracted from nacre and P. radiata flesh to stimulate bone formation.

The effect of nacre extract on cord blood-derived endothelial progenitor cells: A natural stimulus to promote angiogenesis?

Angiogenesis is a critical parameter to consider for the development of tissue-engineered bone substitutes. The challenge is to promote sufficient vascularization in the bone substitute to prevent cell death and to allow its efficient integration. The capacity of nacre extract to restore the osteogenic activity of osteoarthritis osteoblasts has already been demonstrated. However, their angiogenic potential on endothelial progenitor cells (EPCs) was not yet explored. Therefore, the current study aimed at investigating if nacreous molecules affect EPC behavior. The gene and protein expression levels of endothelial cell (EC)-specific markers were determined in EPCs cultivated in presence of a nacre extract (ethanol soluble matrix [ESM] at two concentrations: 100 µg/mL and 200 µg/mL (respectively abbreviated ESM100 and ESM200)). Cell functionality was explored by proangiogenic factors production and in vitro tube formation assay. ESM200 increased the expression of some EC-specific genes. The in vitro tube formation assay demonstrated that ESM200 stimulated tubulogenesis affecting angiogenic parameters. We demonstrated that a stimulation with 200 µg/mL of ESM increased angiogenesis key elements. This in vitro study strongly highlights the proangiogenic effect of ESM. Due to its osteogenic properties, previously demonstrated, ESM could constitute the key element to develop an ideal prevascularized bone substitute. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Nacre, a natural, multi-use, and timely biomaterial for bone graft substitution.

During the past two decades, with a huge and rapidly increasing clinical need for bone regeneration and repair, bone substitutes are more and more seen as a potential solution. Major innovation efforts are being made to develop such substitutes, some having advanced even to clinical practice. It is now time to turn to natural biomaterials. Nacre, or mother-of-pearl, is an organic matrix-calcium carbonate coupled shell structure produced by molluscs. In vivo and in vitro studies have revealed that nacre is osteoinductive, osteoconductive, biocompatible, and biodegradable. With many other outstanding qualities, nacre represents a natural and multi-use biomaterial as a bone graft substitute. This review aims at summarising the current needs in orthopaedic clinics and the challenges for the development of bone substitutes; most of all, we systematically review the physiological characteristics and biological evidence of nacre's effects centred on osteogenesis, and finally we put forward the potential use of nacre as a bone graft substitute. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 662-671, 2017.

Crystallographic control on the substructure of nacre tablets.

Nacre tablets of mollusks develop two kinds of features when either the calcium carbonate or the organic portions are removed: (1) parallel lineations (vermiculations) formed by elongated carbonate rods, and (2) hourglass patterns, which appear in high relief when etched or in low relief if bleached. In untreated tablets, SEM and AFM data show that vermiculations correspond to aligned and fused aragonite nanogloblules, which are partly surrounded by thin organic pellicles. EBSD mapping of the surfaces of tablets indicates that the vermiculations are invariably parallel to the crystallographic a-axis of aragonite and that the triangles are aligned with the b-axis and correspond to the advance of the {010} faces during the growth of the tablet. According to our interpretation, the vermiculations appear because organic molecules during growth are expelled from the a-axis, where the Ca-CO3 bonds are the shortest. In this way, the subunits forming nacre merge uninterruptedly, forming chains parallel to the a-axis, whereas the organic molecules are expelled to the sides of these chains. Hourglass patterns would be produced by preferential adsorption of organic molecules along the {010}, as compared to the {100} faces. A model is presented for the nanostructure of nacre tablets. SEM and EBSD data also show the existence within the tablets of nanocrystalline units, which are twinned on {110} with the rest of the tablet. Our study shows that the growth dynamics of nacre tablets (and bioaragonite in general) results from the interaction at two different and mutually related levels: tablets and nanogranules.

Pearls are self-organized natural ratchets.

Pearls, the most flawless and highly prized of them, are perhaps the most perfectly spherical macroscopic bodies in the biological world. How are they so round? Why are other pearls solids of revolution (off-round, drop, ringed pearl), and yet others have no symmetry (baroque pearls)? We observe that with a spherical pearl the growth fronts of nacre are spirals and target patterns distributed across its surface, and that this is true for a baroque pearl, too, but that in pearls with rotational symmetry spirals and target patterns are found only in the vicinity of the poles; elsewhere the growth fronts are arrayed in ratchet fashion around the equator. We argue that pearl rotation is a self-organized phenomenon caused and sustained by physical forces from the growth fronts, and that rotating pearls are an example--perhaps unique--of a natural ratchet.

Osteogenic differentiation of human bone marrow mesenchymal stem cells in hydrogel containing nacre powder.

Nacre (or mother of pearl) can facilitate bone cell differentiation and can speed up their mineralization. Here we report on the capability of nacre to induce differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) and the production of extracellular matrix. hBM-MSCs were encapsulated in an alginate hydrogel containing different concentrations of powdered nacre and cultured in the same environment until Day 28. Analysis of osteogenic gene expression, histochemistry, second harmonic generation (SHG) microscopy, and Raman scattering spectroscopy were used to characterize the synthesis of the extracellular matrix. In the presence of nacre powder, a significant increase in matrix synthesis from D21 in comparison with pure alginate was observed. Histochemistry revealed the formation of a new tissue composed of collagen fibers in the presence of nacre (immunostaining and SHG), and hydroxyapatite crystals (Raman) in the alginate beads. These results suggest that nacre is efficient in hBM-MSCs differentiation, extracellular matrix production and mineralization in alginate 3D biomaterials.

Subchondral nacre implant in the articular zone of the sheep's knee: a pilot study.

The present study was designed to analyze the intra-articular behaviour of nacre, when implanted in the subchondral bone area in the sheep knee. We implanted nacre blocks in sheep's trochlea by replacing the half of the femoral trochlea (nacre group). For comparison we used complete cartilage resection (resection group) down to the subchondral bone. In the "nacre group", implants were well tolerated without any synovial inflammation. In addition, we observed centripetal regrowth of new cartilage after 3 months. In the "resection group", no chondral regrowth was observed, but, in contrast, a thin layer of fibrous tissue was formed. After 6 months, a new tissue covered the nacre implant formed by an osteochondral regrowth. Nacre, as a subchondral implant, exerts benefic potential for osteochondral repair.

Dynamics of sheet nacre formation in bivalves.

Formation of nacre (mother-of-pearl) is a biomineralization process of fundamental scientific as well as industrial importance. However, the dynamics of the formation process is still not understood. Here, we use scanning electron microscopy and high spatial resolution ion microprobe depth-profiling to image the full three-dimensional distribution of organic materials around individual tablets in the top-most layer of forming nacre in bivalves. Nacre formation proceeds by lateral, symmetric growth of individual tablets mediated by a growth-ring rich in organics, in which aragonite crystallizes from amorphous precursors. The pivotal role in nacre formation played by the growth-ring structure documented in this study adds further complexity to a highly dynamical biomineralization process.

Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: an environmental- and cryo-scanning electron microscopy study.

A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.

Low molecular weight molecules of oyster nacre induce mineralization of the MC3T3-E1 cells.

The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction, and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy, and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite (HA) in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurrence of clusters of spherical particles resembling to HA. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair.

The effect of molecules in mother-of-pearl on the decrease in bone resorption through the inhibition of osteoclast cathepsin K.

This study evaluates the effect of the mother-of-pearl (nacre) organic matrix on mammalian osteoclast activity and on cathepsin K protease. Rabbit osteoclasts were cultured on bovine cortical bone slices in the presence of water-soluble molecules extracted from nacre of the pearl oyster Pinctada margaritifera. Osteoclast resorption activity was determined by quantification of the resorption surface area on bovine bone slices. Papain and cathepsin K, B and L inhibition tests were performed in the presence of the nacre water-soluble extracts. The active crude extract was fractionated by dialysis and reversed-phase high-performance liquid chromatography before electrospray mass spectrometry analysis of inhibitory fractions. The water-soluble molecules extracted from nacre decreased bone resorption without jeopardizing osteoclast survival. The hydrolytic activity of cysteine proteinases was reduced when the enzymes were incubated with the nacre water-soluble molecules. Trending towards characterization of the molecules involved, it appears that cathepsin K inhibitors remain in different nacre water-soluble organic matrix subfractions, composed of low molecular weight molecules. Mollusk shell nacre contains molecules capable of reducing osteoclast bone resorption activity by inhibiting cathepsin K, giving a new facet of the bioactivity of nacre as bone biomaterial.

Proteomics analysis of the nacre soluble and insoluble proteins from the oyster Pinctada margaritifera.

Shell nacre is laid upon an organic cell-free matrix, part of which, paradoxically, is water soluble and displays biological activities. Proteins in the native shell also constitute an insoluble network and offer a model for studying supramolecular organization as a means of self-ordering. Consequently, difficulties are encountered in extraction and purification strategies for protein characterization. In this work, water-soluble proteins and the insoluble conhiolin residue of the nacre of Pinctada margaritifera matrix were analyzed via a proteomics approach. Two sequences homologous to nacre matrix proteins of other Pinctada species were identified in the water-soluble extract. One of them is known as a fundamental component of the insoluble organic matrix of nacre. In the conchiolin, the insoluble residue, four homologs of Pinctada nacre matrix proteins were found. Two of them were the same as the molecules characterized in the water-soluble extract. Results established that soluble and insoluble proteins of the nacre organic matrix share constitutive material. Surprisingly, a peptide in the conchiolin residue was found homologous to a prismatic matrix protein of Pinctada fucata, suggesting that prismatic and nacre matrices may share common proteins. The insoluble properties of shell matrix proteins appear to arise from structural organization via multimerization. The oxidative activity, found in the water-soluble fraction of the nacre matrix, is proposed as a leading process in the transformation of transient soluble proteins into the insoluble network of conchiolin during nacre growth.

Heterogeneity of proteinase inhibitors in the water-soluble organic matrix from the oyster nacre.

We extracted proteinase inhibitors from the nacre of the oyster Pinctada margaritifera with water. Mixing the nacre powder with water for 20 h led to a water-soluble fraction [0.24% (wt/wt) of nacre]. After dialysis of the water-soluble matrix through 6- to 8-kDa and 0.5-kDa membranes, the proteinase inhibitors were divided into low and high molecular weight fractions that contained inhibitors of papain, bovine cathepsin B, and human cathepsin L. We studied the heterogeneity of the inhibitors after separating the low molecular weight fraction according to charge and hydrophobicity. After multistep purification, mass spectrometry analysis revealed that a potent inhibitory fraction contained several molecules. This observation demonstrates the difficulties encountered in attempting to isolate individual metabolites from the complex mixture of molecules present in nacre matrix. Interestingly, the low molecular weight fraction contained specific inhibitors that could discern between cathepsin B and cathepsin L. The nacre organic inhibitors were active against several cysteine proteinases, yet they were more specific in relation to serine proteinases, because only proteinase K was inhibited. These results demonstrate, for the first time, the presence of active proteinase inhibitors in the mollusc shell, and it is possible that these inhibitors may play a role in either protection of proteins involved in shell formation or in defense against parasites, or both.