Biomineral-Based Composite Materials in Regenerative Medicine.

Regenerative medicine aims to address substantial defects by amplifying the body's natural regenerative abilities and preserving the health of tissues and organs. To achieve these goals, materials that can provide the spatial and biological support for cell proliferation and differentiation, as well as the micro-environment essential for the intended tissue, are needed. Scaffolds such as polymers and metallic materials provide three-dimensional structures for cells to attach to and grow in defects. These materials have limitations in terms of mechanical properties or biocompatibility. In contrast, biominerals are formed by living organisms through biomineralization, which also includes minerals created by replicating this process. Incorporating biominerals into conventional materials allows for enhanced strength, durability, and biocompatibility. Specifically, biominerals can improve the bond between the implant and tissue by mimicking the micro-environment. This enhances cell differentiation and tissue regeneration. Furthermore, biomineral composites have wound healing and antimicrobial properties, which can aid in wound repair. Additionally, biominerals can be engineered as drug carriers, which can efficiently deliver drugs to their intended targets, minimizing side effects and increasing therapeutic efficacy. This article examines the role of biominerals and their composite materials in regenerative medicine applications and discusses their properties, synthesis methods, and potential uses.

Insight into biomolecular interaction-based non-classical crystallization of bacterial biocement.

In an attempt to draw a correlation between calcium carbonate (CaCO3) precipitation and biomacromolecules such as extracellular polymeric substances and enzyme activity in biomineralizing microbe, this report aims to elucidate the ureolytic and ammonification route in Paenibacillus alkaliterrae to explore the possible role of organic biomolecule(s) present on cell surface in mediating nucleation and crystallization of biogenic CaCO3. After 168 h of biomineralization in ureolysis and ammonification, 2.2 g/l and 0.87 g/l of CaCO3 precipitates were obtained, respectively. The highest carbonic anhydrase activity (31.8 µmoles/min/ml) was evidenced in ammonification as opposed to ureolysis (24.8 µmoles/min/ml). Highest urease activity reached up to 9.26 µmoles/min/ml in ureolytic pathway. Extracellular polymeric substances such as polysaccharides and proteins were found to have a vital role not only in the nucleation and crystal growth but also in addition direct polymorphic fate of CaCO3 nanoparticles. EPS production was higher during ammonification (3.1 mg/ml) than in ureolysis (0.72 mg/ml). CaCO3 nanoparticle-associated proteins were found to be 0.82 mg/ml in ureolysis and 0.56 mg/ml in ammonification. After 30 days of biomineralization, all the polymorphic forms stabilized to calcite in ureolysis but in ammonification vaterite predominated. In our study, we showed that organic template-mediated prokaryotic biomineralization follows the non-classical nucleation and varying proportions of these organic components causes selective polymorphism of CaCO3 nanoparticles. Overall, the findings are expected to further the fundamental understanding of enzymes, EPS-driven non-classical nucleation of CaCO3, and we foresee the design of fit-for-purpose futuristic biominerals arising from such renewed understanding of biomineralization. KEY POINTS: • Organic-inorganic interface of cell surface promote crystallization of biominerals • Carbohydrate and proteins in the interface results selective polymorphism of CaCO3 • Calcite stabilized at 30 days in ureolysis, vaterite-calcite mix in ammonification.

From particle attachment to space-filling coral skeletons.

Reef-building corals and their aragonite (CaCO3) skeletons support entire reef ecosystems, yet their formation mechanism is poorly understood. Here we used synchrotron spectromicroscopy to observe the nanoscale mineralogy of fresh, forming skeletons from six species spanning all reef-forming coral morphologies: Branching, encrusting, massive, and table. In all species, hydrated and anhydrous amorphous calcium carbonate nanoparticles were precursors for skeletal growth, as previously observed in a single species. The amorphous precursors here were observed in tissue, between tissue and skeleton, and at growth fronts of the skeleton, within a low-density nano- or microporous layer varying in thickness from 7 to 20 µm. Brunauer-Emmett-Teller measurements, however, indicated that the mature skeletons at the microscale were space-filling, comparable to single crystals of geologic aragonite. Nanoparticles alone can never fill space completely, thus ion-by-ion filling must be invoked to fill interstitial pores. Such ion-by-ion diffusion and attachment may occur from the supersaturated calcifying fluid known to exist in corals, or from a dense liquid precursor, observed in synthetic systems but never in biogenic ones. Concomitant particle attachment and ion-by-ion filling was previously observed in synthetic calcite rhombohedra, but never in aragonite pseudohexagonal prisms, synthetic or biogenic, as observed here. Models for biomineral growth, isotope incorporation, and coral skeletons' resilience to ocean warming and acidification must take into account the dual formation mechanism, including particle attachment and ion-by-ion space filling.

Novel Focused Ion Beam Liftouts for Spatial Characterization of Spherical Biominerals With Transmission Electron Microscopy.

Focused ion beam (FIB) is frequently used to prepare electron- and X-ray-beam-transparent thin sections of samples, called lamellae. Typically, lamellae are prepared from only a subregion of a sample. In this paper, we present a novel approach for FIB lamella preparation of microscopic samples, wherein the entire cross-section of the whole sample can be investigated. The approach was demonstrated using spherical, porous, and often hollow microprecipitates of biologically precipitated calcium carbonate. The microprecipitate morphology made these biogenic samples more fragile and challenging than materials commonly investigated using FIB lamellae. Our method enables the appropriate orientation of the lamellae required for further electron/X-ray analyses after attachment to the transmission electron microscopy (TEM) grid post and facilitates more secure adhesion onto the grid post. We present evidence of autofluorescence in bacterially precipitated vaterite using this lamella preparation method coupled with TEM selected area diffraction. This innovative approach allows studying biomineralization at the micro to nano scales, which can provide novel insights into bacterial responses to microenvironmental conditions.

Review on the management of water quality for bio-mineral swimming pools in Western Europe.

In this review, we depict the state of the art concerning the water quality management of bio-mineral bathing pools, and compare these to traditional swimming pools. Bio-mineral pools use a combination of mechanic filtration, bio-filtration, and UV-treatment to disinfect the water. Studies in test tanks have shown that bio-filtration is effective in maintaining the water quality with regard to the treatment of organic pollution. Concerning biological risks, the bio-mineral pool relies on UV-treatment to degrade bacteria. Unlike chemical disinfectant treatments, UV disinfection does not lose its effectiveness in the event of high traffic in the pool. However, as only the water taken up by the filtration system is disinfected, it is essential that all the water in the pool is filtered. If the pool has a dead zone, its water is not disinfected and there is a risk of localized pathogen development. As the development of bio-mineral pools spreads in Europe, legislation gradually follows. The health parameters measured differ slightly from one country to another, but there are constants: the measurement of Escherichia coli, Enterococci, and Pseudomonas aeruginosa. In terms of biological swimming pools, regulatory homogeneity across Europe does not exist. From these comparisons, Austrian legislation segmenting water quality into 4 categories ranging from "excellent" to "poor" represents legislation that combines health and safety with indications of possible malfunctions. Next, a study of three real sites of bio-mineral pools is presented. It appears that whatever the type of pool, bio-mineral filtration makes it possible to achieve performances comparable to those encountered in chlorinated swimming pools concerning the risks associated with fecal contamination and external pollution. On the other hand, when frequentation is high, as is the case in small pools used for aquafitness, monitoring the risks of inter-bather contamination, as illustrated by the presence of Staphylococcus aureus, reveals a recurring problem. Knowing that this parameter is not evaluated in bathing waters in the natural environment and that numerous studies show that Staphyloccocus aureus are always detected, even on beaches, we propose the definition of three thresholds: i.e., 0 CFU/100 mL (threshold value in Wallonia) for water of excellent quality, less than 20 CFU/100 mL (threshold value in France) for water of very good quality, less than 50 CFU/100 mL (contribution of bathers by simple immersion) for good quality water, and more than 50 CFU/100 mL for poor quality water. This document could therefore be converted into a manual for operators on the use and management of bio-mineral baths.

Selenium-doped calcium phosphate biomineral reverses multidrug resistance to enhance bone tumor chemotherapy.

The construction of a functional drug delivery system to reverse the multidrug resistance (MDR) of bone tumors in cases of failed chemotherapy remains a challenge. Herein, we demonstrate a selenium-doped calcium phosphate (Se-CaP) biomineral with high biocompatibility, biodegradability and pH-sensitive drug release properties. Se-CaP may not only serve as an effective drug-carrier to enhance the uptake of doxorubicin (DOX), but may also synchronously induce caspases-mediated apoptosis of osteosarcoma by generating intracellular reactive oxygen species (ROS). Furthermore, in vitro and in vivo studies obviously demonstrate that Se-CaP can reverse the MDR of osteosarcoma by down-regulating the expression of MDR-related ABC (ATP binding cassette) transporters proteins (ABCB1 and ABCC1). Finally, DOX-loaded Se-CaP can significantly inhibit DOX-resistant MG63 (MG63/DXR) tumor growth in nude mice. Considering its biomimetic chemical properties, the Se-CaP biomineral, with the multiple functions mentioned above, could be a promising candidate for treating bone tumors with MDR characteristics.

Mineralogy, geochemistry, 13C and 16O isotopic characteristics of urinary stones in Iran, a case study of Lorestan Province.

Owing to the importance of urinary stones as one of the biominerals in the human body, it is necessary to investigate their chemical composition and mineralogy. In this matter, a mineralogical study using X-ray diffraction and scanning electron microscopy indicated that urinary stones in Lorestan Province were divided into 5 groups of calcium oxalate, urate, cysteine, phosphate and mixed stones (Whewellite, uric acid, phosphate). In this regard, the microscopic studies revealed that Whewellite was the most important mineral phase among various phases. In the following, the major and rare elements of each group were determined by inductively coupled plasma mass spectrometry (ICP-MS) and X-ray fluorescence analysis. The obtained results demonstrated that Ca was found the most abundant element in urinary stones. In the analysis results of the major oxides, compared to other major oxides, CaO had the highest frequency in urinary stones. The reason was due to the role of calcium in most of the basic functions in cell metabolism. The average values of isotope 13C and 16O in the studied urinary stones were obtained - 33.71 and - 20.57, respectively. Overall, the values of 13C isotope in urinary stones were lower than those in the similar stones and human hard tissues in other countries.

Mollusc shellomes: Past, present and future.

In molluscs, the shell fabrication requires a large array of secreted macromolecules including proteins and polysaccharides. Some of them are occluded in the shell during mineralization process and constitute the shell repertoire. The protein moieties, also called shell proteomes or, more simply, 'shellomes', are nowadays analyzed via high-throughput approaches. These latter, applied so far on about thirty genera, have evidenced the huge diversity of shellomes from model to model. They also pinpoint the recurrent presence of functional domains of diverse natures. Shell proteins are not only involved in guiding the mineral deposition, but also in enzymatic and immunity-related functions, in signaling or in coping with many extracellular molecules such as saccharides. Many shell proteins exhibit low complexity domains, the function of which remains unclear. Shellomes appear as self-organizing systems that must be approached from the point of view of complex systems biology: at supramolecular level, they generate emergent properties, i.e., microstructures that cannot be simply explained by the sum of their parts. A conceptual scheme is developed here that reconciles the plasticity of the shellome, its evolvability and the constrained frame of microstructures. Other perspectives arising from the study of shellomes are briefly discussed, including the macroevolution of shell repertoires, their maturation and their transformation through time.

Microstructural and compositional variation in pacu and piranha teeth related to diet specialization (Teleostei: Serrasalmidae).

In any vertebrate group, tooth shape is known to fit with a biological function related to diet. However, little is known about the relationships between diet and tooth microstructure and composition in teleost fishes. In this work, we describe the external morphology, internal microstructure and elemental composition of the oral teeth of three representative species of the family Serrasalmidae having different feeding habits (herbivorous vs. omnivorous vs. carnivorous). We used backscattered-electron imaging and low vacuum environmental scanning electron microscope to compare the organization and mineralization of tooth layers as well as energy dispersive X-ray microanalysis and Raman microspectrometry to investigate the elemental composition, Ca/P ratio and mineralogy of the most superficial layers. Oral teeth of each serrasalmid species have the same internal organization based on five distinctive layers (i.e. pulp, dentine, inner enameloid, outer enameloid and cuticle) but the general tooth morphology is different according to diet. Microstructural and compositional variation of the cuticle and iron-enrichment of superficial layers were highlighted between herbivorous and carnivorous species. Iron is more concentrated in teeth of the herbivorous species where it is associated with a thicker cuticle explaining the more intense red-pigmentation of the cutting edges of oral teeth. The iron-enrichment is interpreted as a substitution of Ca by Fe in the hydroxyapatite. These traits are discussed in the light of the evolutionary history of the family. Further considerations and hypotheses about the formation and origin of the mineralized tooth layers and especially the iron-rich superficial layers in teleost fishes are suggested.

Biopolymer Stabilization/Solidification of Soils: A Rapid, Micro-Macro, Cross-Disciplinary Approach.

In this study, we describe a novel high throughput, micro-macro approach for the identification and efficient design of biopolymer stabilized soil systems. At the "microscopic" scale, we propose a rapid Membrane Enabled Bio-Mineral Affinity Screening (MEBAS) approach supported by Mineral Binding Characterization (MBC) (TGA, ATR-FTIR and ζ Potential), while at the "macroscopic" scale, micro scale results are confirmed by Geotechnical Verification (GV) through unconfined compression testing. We illustrate the methodology using an exemplar mine tailings Fe2O3-SiO2 system. Five different biopolymers were tested against Fe2O3: locust bean gum, guar gum, gellan gum, xanthan gum, and sodium carboxymethyl cellulose. The screening revealed that locust bean gum and guar gum have the highest affinity for Fe2O3, which was confirmed by MBC and in agreement with GV. This affinity is attributed to the biopolymer's ability to form covalent C-O-Fe bonds through ß-(1,4)-d-mannan groups. Upon their 1% addition to a "macroscopic" Fe2O3 based exemplar MT system, unconfined compressive strengths of 5171 and 3848 kPa were obtained, significantly higher than those for the other biopolymers and non-Fe systems. In the current study, MEBAS gave an approximately 50-fold increase in rate of assessment compared to GV alone. Application of the proposed MEBAS-MBC-GV approach to a broad range of soil/earthwork components and additives is discussed.

Biological Kerker Effect Boosts Light Collection Efficiency in Plants.

Being the polymorphs of calcium carbonate (CaCO3), vaterite and calcite have attracted a great deal of attention as promising biomaterials for drug delivery and tissue engineering applications. Furthermore, they are important biogenic minerals, enabling living organisms to reach specific functions. In nature, vaterite and calcite monocrystals typically form self-assembled polycrystal micro- and nanoparticles, also referred to as spherulites. Here, we demonstrate that alpine plants belonging to the Saxifraga genus can tailor light scattering channels and utilize multipole interference effect to improve light collection efficiency via producing CaCO3 polycrystal nanoparticles on the margins of their leaves. To provide a clear physical background behind this concept, we study optical properties of artificially synthesized vaterite nanospherulites and reveal the phenomenon of directional light scattering. Dark-field spectroscopy measurements are supported by a comprehensive numerical analysis, accounting for the complex microstructure of particles. We demonstrate the appearance of generalized Kerker condition, where several higher order multipoles interfere constructively in the forward direction, governing the interaction phenomenon. As a result, highly directive forward light scattering from vaterite nanospherulites is observed in the entire visible range. Furthermore, ex vivo studies of microstructure and optical properties of leaves for the alpine plants Saxifraga "Southside Seedling" and Saxifraga Paniculata Ria are performed and underline the importance of the Kerker effect for these living organisms. Our results pave the way for a bioinspired strategy of efficient light collection by self-assembled polycrystal CaCO3 nanoparticles via tailoring light propagation directly to the photosynthetic tissue with minimal losses to undesired scattering channels.

Manganese enhances the immobilization of trace cadmium from irrigation water in biological soil crust.

The effect of biological soil crust (BSC) in paddy field on the immobilization and removal of heavy metal from irrigation water is an important issue. BSC was cultured in solutions with different concentrations of manganese (Mn) salt and cadmium (Cd) sulfate for 15 days. We analyzed the Mn, Cd and Fe contents in the BSC and investigated the effects of Mn salt on the Cd distribution in different binding-forms in BSC as well. The results show that Mn salt was effective at enabling BSC to immobilize the Cd, and its removal efficiency from irrigation water improved with an increase in the Mn concentration used. The removal of 50.00 µg/L of Cd from irrigation water by BSC reached as high as 95.70% in present of 20.00 mg/L Mn. The highest obtained biological concentrated factor of BSC for Cd is ~2.7 × 104. The mainly Cd species (75%) in BSC is the non-EDTA extracted minerals. Based on the SEM-EDS and XPS analyses, it was reasonably inferred that the Mn ion was oxidized by Mn oxidizing bacteria (MOB), to yield the porous spongy-like birnessite with d-spacing of 2.31 Ǻ, while Cd was scavenged and immobilized in the crystal lattice. The MOB was identified as Bacillus. This study provides a potentially novel method to decontaminate irrigation water polluted with Cd by using BSC in presence of Mn.

Enzymatic Reaction Generates Biomimic Nanominerals with Superior Bioactivity.

In vivo mineralization is a multistep process involving mineral-protein complexes and various metastable compounds in vertebrates. In this complex process, the minerals produced in the mitochondrial matrix play a critical role in initiating extracellular mineralization. However, the functional mechanisms of the mitochondrial minerals are still a mystery. Herein, an in vitro enzymatic reaction strategy is reported for the generation of biomimic amorphous calcium phosphate (EACP) nanominerals by an alkaline phosphatase (ALP)-catalyzed hydrolysis of adenosine triphosphate (ATP) in a weakly alkalescent aqueous condition (pH 8.0-8.5), which is partially similar to the mitochondrial environment. Significantly, the EACP nanomineral obviously promotes autophagy and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by activating an AMPK related pathway, and displays a high performance in promoting bone regeneration. These results provide in vitro evidence for the effect of ATP on the formation and stabilization of the mineral in the mineralization process, demonstrating a potential strategy for the preparation of the biomimic mineral for treating bone related diseases.

The Ferritin Superfamily.

Iron is very important in many biological processes and the ferritin protein family has evolved to store iron and to maintain cellular iron homeostasis. The deletion of the coding gene for the H subunit of ferritin leads to early embryonic death in mice and mutations in the gene for the L subunits in humans has been observed in neurodegenerative diseases, such as neuroferritinopathy. Thus, understanding how ferritin works is imperative and many studies have been conducted to delineate the molecular mechanism of ferritins and bacterioferritins. In the ferritin protein family, it is clear that a catalytic center for iron oxidation, the routes for iron to reach this center and the ability to nucleate an iron core, are common requirements for all ferritins. However, there are differences in the structural and mechanistic details of iron oxidation and mineralization. Although a common mechanism has been proposed for all ferritins, this mechanism needs to be further explored. There is a mechanistic diversity related to structural variation in the ferritin protein family. It is clear that other factors appear to affect the mechanism of iron oxidation and mineralization. This review focusses on the structural features of the ferritin protein family and its role in the mechanism of iron mineralization.

Glassin, a histidine-rich protein from the siliceous skeletal system of the marine sponge Euplectella, directs silica polycondensation.

The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure-function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named "glassin," the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6-8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.

The Understanding of the Metazoan Skeletal System, Based on the Initial Discoveries with Siliceous and Calcareous Sponges.

Initiated by studies on the mechanism of formation of the skeletons of the evolutionary oldest still extant multicellular animals, the sponges (phylum Porifera) have provided new insights into the mechanism of formation of the Ca-phosphate/hydroxyapatite skeleton of vertebrate bone. Studies on the formation of the biomineral skeleton of sponges revealed that both the formation of the inorganic siliceous skeletons (sponges of the class of Hexactinellida and Demospongiae) and of the calcareous skeletons (class of Calcarea) is mediated by enzymes (silicatein: polymerization of biosilica; and carbonic anhydrase: deposition of Ca-carbonate). Detailed studies of the initial mineralization steps in human bone-forming cells showed that this process is also controlled by enzymes, starting with the deposition of Ca-carbonate bio-seeds, mediated by carbonic anhydrases-II and -IX, followed by non-enzymatic transformation of the formed amorphous Ca-carbonate deposits into amorphous Ca-phosphate and finally hydroxyapatite crystals. The required phosphate is provided by enzymatic (alkaline phosphatase-mediated) degradation of an inorganic polymer, polyphosphate (polyP), which also acts as a donor for chemically useful energy in this process. These new discoveries allow the development of novel biomimetic strategies for treatment of bone diseases and defects.

Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation.

Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3 (-) ) cause induction of expression of the CA in human osteogenic SaOS-2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, vaterite, and aragonite, is significantly accelerated in the presence of a peptide rich in aspartic acid and glutamic acid. We propose that the initial CaCO3 bioseed formation is driven by CA, and that the subsequent conversion to calcium phosphate/calcium hydroxyapatite (exchange of carbonate by phosphate) is a non-enzymatic exchange process.

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.

Limitations and modifications in the clinical application of calcium sulfate.

Calcium sulfate and calcium sulfate-based biomaterials have been widely used in non-load-bearing bone defects for hundreds of years due to their superior biocompatibility, biodegradability, and non-toxicity. However, lower compressive strength and rapid degradation rate are the main limitations in clinical applications. Excessive absorption causes a sharp increase in sulfate ion and calcium ion concentrations around the bone defect site, resulting in delayed wound healing and hypercalcemia. In addition, the space between calcium sulfate and the host bone, resulting from excessively rapid absorption, has adverse effects on bone healing or fusion techniques. This issue has been recognized and addressed. The lack of sufficient mechanical strength makes it challenging to use calcium sulfate and calcium sulfate-based biomaterials in load-bearing areas. To overcome these defects, the introduction of various inorganic additives, such as calcium carbonate, calcium phosphate, and calcium silicate, into calcium sulfate is an effective measure. Inorganic materials with different physical and chemical properties can greatly improve the properties of calcium sulfate composites. For example, the hydrolysis products of calcium carbonate are alkaline substances that can buffer the acidic environment caused by the degradation of calcium sulfate; calcium phosphate has poor degradation, which can effectively avoid the excessive absorption of calcium sulfate; and calcium silicate can promote the compressive strength and stimulate new bone formation. The purpose of this review is to review the poor properties of calcium sulfate and its complications in clinical application and to explore the effect of various inorganic additives on the physicochemical properties and biological properties of calcium sulfate.