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.

Biochemical and thermodynamic characterization of mutated ß1,4-galactosyltransferase 7 involved in the progeroid form of the Ehlers-Danlos syndrome.

Three mutations of the B4GALT7 gene [encoding ß1,4-GalT7 (ß1,4-galactosyltransferase 7)], corresponding to A186D, L206P and R270C, have been identified in patients with the progeroid form of the Ehlers-Danlos syndrome and are described as being associated with the reduction or loss of ß1,4-GalT7 activity. However, the molecular basis of the reduction or loss of activity remained to be determined. In the present study, wild-type, A186D, L206P and R270C ß1,4-GalT7 were expressed in CHO618 cells as membrane proteins and in Escherichia coli as soluble proteins fused to MBP (maltose-binding protein). The ability of the expressed proteins to transfer galactose from donor to acceptor substrates was systematically characterized by kinetic analysis. The physicochemical properties of soluble proteins were explored by isothermal titration calorimetry, which is a method of choice when determining the thermodynamic parameters of the binding of substrates. Together, the results showed that: (i) the L206P mutation abolished the activity when L206P ß1,4GalT7 was either inserted in the membrane or expressed as a soluble MBP-full-length fusion protein; (ii) the A186D mutation weakly impaired the binding of the donor substrate; and (iii) the R270C mutation strongly impaired the binding of the acceptor substrate. Moreover, the ex vivo consequences of the mutations were investigated by evaluating the priming efficiency of xylosides on GAG (glycosaminoglycan) chain initiation. The results demonstrate a quantitative effect on GAG biosynthesis, depending on the mutation; GAG biosynthesis was fully inhibited by the L206P mutation and decreased by the R270C mutation, whereas the A186D mutation did not affect GAG biosynthesis severely.

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.

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.

Metabolism of parabens (4-hydroxybenzoic acid esters) by hepatic esterases and UDP-glucuronosyltransferases in man.

Parabens (alkyl esters of 4-hydroxybenzoic acid) are widely used as preservatives in drugs, cosmetic products, and foodstuffs. Safety concerns have recently increased due to the potential health risks associated to exposure to large amounts of these substances. Biotransformation of parabens mainly includes hydrolysis of the ester bond and glucuronidation reactions. The hydrolysis and glucuronidation of a series of six parabens differing by the nature of the alkyl group were investigated in human liver microsomes and plasma, and the major human UDP-glucuronosyltransferase (UGT) isoforms involved in the reaction were identified. Methyl- and ethylparaben were stable in human plasma, with 95% of the initial concentration remaining after 24 h. On the other hand, propyl-, butyl- and benzylparaben concentrations decreased by 50% under similar conditions. In contrast, rapid hydrolysis was measured with human liver microsomes depending on the alkyl chain length, with t(1/2) varying from 22 min for methylparaben to 87 min for butylparaben. All parabens were actively glucuronidated by liver microsomes, in comparison to 4-hydroxybenzoic acid. They were mainly substrates of human recombinant UGT1A1, UGT1A8, UGT1A9, UGT2B7, UGT2B15 and UGT2B17. In conclusion, the parabens were readily metabolized in human liver through esterase hydrolysis and glucuronidation by several UGT isoforms. These results suggest that these parabens do not accumulate in human tissue.

Characterization of catechol glucuronidation in rat liver.

Catechols are a class of substances from natural or synthetic origin that contain a 1,2-dihydroxybenzene group. We have characterized the glucuronidation by rat liver microsomes and by the rat liver recombinant UDP-glucuronosyltransferase isoforms UGT1A6 and UGT2B1 of a series of 42 structurally diverse catechols, including neurotransmitters, polyphenols, drugs, and catechol estrogens. Small catechols (4-nitrocatechol, 2,3-dihydroxybenzaldehyde, 4-methylcatechol, and tetrachlorocatechol), tyrphostine A23, and octylgallate were glucuronidated at the highest rate by rat liver microsomes and the recombinant enzymes. By contrast, polyphenols from green tea (catechin and related compounds), 3,5-dinitrocatechol, the catechol-O-methyltransferase inhibitor drugs (entacapone, nitecapone, and tolcapone), the carboxyl catechols (gallic acid and dihydroxybenzoic acid derivatives), and the neurotransmitters and dopaminergic drugs, except dobutamine, were glucuronidated at low rate. Glucuronidation of most catechols was increased upon treatment of rats by 3-methylcholanthrene (3-MC) or Aroclor 1254. No induction was observed after administration of phenobarbital and clofibrate or treatment with catechols. Partial least-squares modeling was carried out to explain the variations of glucuronidation activity by liver microsomes of nontreated and 3-MC-treated rats. The model developed explained 82% and predicted 61% of the variations of glucuronidation activities. Among the 17 electronic and substructure parameters used that characterize the catechols, the hydrophobicity/molar volume ratio of catechols showed a strong positive correlation with the glucuronidation rate. The effect of the pK(a) of the catechol group was modeled to be nonlinear, the optimal pK(a) value for glucuronidation being between 8 and 9. Hydrogen bonding and steric effects also were important to account for to predict the glucuronidation rates.