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Kalcijev karbonat (CaCO3) je jedan od najrasprostranjenijih minerala u morskom okolišu. Nastaje procesima taloženja pod kojima podrazumjevamo procese formiranja nove čvrste faze iz homogenog sustava, preciznije iz vodenih, obično elektrolitnih otopina, kao što je slučaj i u biološkim sustavima. Procesi taloženja su fizikalno-kemijska osnova biomineralizacije (procesi formiranja biominerala u biološki sustavima). Biomineralizacijom u kalcificirajućim organizmima (mekušcima, koraljima, spužvama, krednjacima...) nastaje biogeni CaCO3. Glavna pokretačka sila koja kontrolira procese taloženja jest prezasićenost, no i drugi faktori kao što su koncentracija konstituirajućih iona i otopljenog ugljikovog dioksida (CO2), prisutnost aditiva, temperatura (t), pH, ionska jakost, hidrodinamika kontroliraju taloženje CaCO3 te posljedično i biomineralizaciju.

Gallic acid (3, 4, 5-trihydroxybenzoic acid) is a secondary polyphenolic metabolite and natural antioxidant mostly found in plants and foodstuff (e.g. strawberry, blueberry, plums, walnut, tea etc.). It shows antioxidative, anticancer and antibacterial activity [1]. Gallic acid is often used in pharmaceutical industry and herbal medicine as well as food supplement [2]. It can also be used as a potential inhibitor of calcium oxalate precipitation [3]. The main goal of our study was to use voltammetric methods for detection of gallic acid in calcium oxalate system. Calcium oxalate was prepared by spontaneous precipitation after mixing calcium solution and oxalate solution in the presence of gallic acid as additive. Calcium oxalate precipitates and calcium oxalate supernatant solution were isolated for electrochemical detection of gallic acid. Electrochemical measurements were conducted in a three electrode voltammetric cell: working glassy carbon electrode, counter electrode platinum wire and Ag/AgCl reference electrode in a phosphate buffer at three different pH values (pH = 5.8, 6.5 and 7.5). The system was purged with high purity argon before each measurement. Differential pulse voltammetry was used to detect gallic acid in calcium oxalate supernatant solution. Measurements in model systems (only gallic acid solution) revealed linear response in a concentration range from 80 mol dm-3 to 1.75 mmol dm-3. The obtained results indicated adsorption/incorporation of gallic acid in the calcium oxalate precipitates, regardless of the pH values. Cyclic voltammetry was used to detect interactions between gallic acid and calcium oxalate precipitates.

Urolithiasis, the formation of urinary stones in different parts of kidney or bladder, is a specific form of pathological biomineralization. Recently, an increasing prevalence of kidney stones in kidney stones in industrial countries is observed and the interest of scientists to explain their pathogenesis with a special focus on calcium oxalate stones is renewed. Calcium oxalates crystallize in the form of hydrated salts: thermodynamically stable calcium oxalate monohydrate (COM, CaC2O4·H2O), metastable dihydrate (COD, CaC2O4·2H2O) and trihydrate (COT, CaC2O4·3H2O). The kidney stones formation under biological conditions can be triggered by various metabolic disorders such as: hypercalciuria, hypocitraturia, hyperoxaluria and the change in the urine acidity. The mechanisms and the conditions under which they crystallize are still not completely clarified. In this work, the spontaneous precipitation and characterization of calcium oxalate monohydrate under conditions of hiperoxaluria (ci(Ca2+) = 7.5 ∙ 10-3 mol dm-3 and ci(C2O42-) = 6.0 ∙ 10-3 mol dm-3) is reported. The experiments were conducted in a model system (Ic = 0.3 mol dm-3 NaCl, which imitates the physiological conditions in the human body) at two initial pH (pHi = 5.0 and 9.0) and with the addition of amino acids reportedly important for pathologic biomineralization. The amino acids selected for the addition are often found in the urine of healthy people and in the organic matrix which is an integral part of kidney stones. The reactant solutions were mixed under controlled hydrodynamic and thermodynamic conditions. Changes in the structure and morphology of precipitated calcium oxalate monohydrate were observed by means of PXRD, SEM, IR and TGA.

According to the latest epidemiological studies, the prevalence of urolithiasis has been increasing for the past few decades, especially in industrialised countries, possibly due to improved standard of living and modern dietary habits. Urolithiasis is the formation of crystals in the urinary tract and is a specific form of pathological biomineralisation. It is a result of physiochemical mechanisms and involves the processes of nucleation, crystal growth and aggregation. Many factors may contribute to crystallisation and urinary stone formation including pH, ionic strength and the presence or absence of substances that can promote or inhibit the process [1]. Different metabolic disorders, which can be inherited or developed, are considered to be important risk factors and include hyperoxaluria, hypercalciuria, hypocitraturia and changes in acidity of the urine. Hyperoxaluria is associated with oxalate urinary tract stones and oxalate crystal deposition in tissue [2]. In this work, systematic research of spontaneous calcium oxalate precipitation in three systems with different levels of complexity (simple, NaCl and artificial urine system) and two different initial pHs (pHi = 5.0 and 9.0) was conducted. In all investigated systems which simulate the conditions of hyperoxaluria, a dominant precipitation of calcium oxalate monohydrate (COM) was noted, except in the artificial urine system at pHi = 9.0, where precipitation of a mixture of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) occurred. The most pronounced difference was observed in COM morphology. In the simple system crystals precipitated in dendritic form, in the NaCl system they took a dendritic form with more or less irregular shaped crystals, while in artificial urine aggregated and prismatic hexagonal COM crystals precipitated.

Calcium carbonate (CaCO3) is an abundant mineral ; it makes up approximately 5 wt% of the Earth's crust, [1] but it can also be found in organisms and biological systems, like mollusks and egg shells, pearls, corals and exoskeletons of arthropods.[2] Calcium carbonate precipitates in the form of several distinct solid phases: amorphous calcium carbonate (ACC), two hydrates (monohydrate and hexahydrate), and three anhydrous polymorphs (calcite, aragonite and vaterite) of which calcite is the most stable form under standard conditions. Aragonite occurs mainly in skeletons of mollusks and corals, in pearls and in hot springs' sediment.[3, 4] Magnesium ions and temperature are two key parameters which control the aragonite formation. In this study, the influence of Mg2+ and temperature on the phase composition, aggregation and morphology of precipitated calcium carbonate crystals is investigated. However, the supersaturation was also considered as a critical parameter of precipitation. It was found that in the low- supersaturation system, at increased Mg2+ concentration and at increased temperature, the aragonite crystals are less aggregated. In addition the amount of crystals sharing {; ; 110}; ; faces and having larger extension of {; ; 001}; ; faces increases. On the contrary, in the high- supersaturation system formation of high magnesium calcite was observed. The obtained results may be relevant in biological, geochemical and technological contexts. [1] W. D. Carlson, Rev. Mineral., 1983, 11, 191–225. [2] G. Falini and S. Fermani, Cryst. Res. Technol., 2013, 48, 864–876. [3] G. Falini, S. Fermani and S. Goffredo, Semin. Cell Dev. Biol., 2015, 46, 17–26. [4] C. Perdikouri, A. Kasioptas, T. Geisler, B. C. Schmidt and A. Putnis, Geochim. Cosmochim. Acta, 2011, 75, 6211–6224.

Main focus of presented investigations are precipitation processes of CaCO3, specifically in the context of biomineralization. To investigate these processes poly aspartic acid and poly glutamic acid were used as synthetic analogues of the natural acidic proteins present in biominerals. Inhibition of CaCO3 precipitation and change of morphology of CaCO3 that we observed when polyaspartic and poly glutamic acid were added into the precipitation systems are direct consequence of a strong adsorption of the chosen acidic polypeptides to the crystal surface.

The preparation of new materials with superior mechanical properties has long been a challenge for materials scientists. Increasingly, materials scientists are focusing on emulating biomineralization processes in so-called biomimetic approaches where the principles of controlled crystal growth in living organisms are applied in the laboratory with the aim of synthesising new materials, with enhanced physical-chemical or mechanical properties. Molluscs’ shells are example of natural biocomposites in which calcium carbonate (CaCO3) is the principal mineral component. The most extensively deposited CaCO3 biominerals are calcite and aragonite, of which calcite is thermodynamically the most stable polymorph. Vaterite, the least stable polymorph of CaCO3, is not as wide-spread in nature as the other two polymorphs are. Its occurrence in nature is often associated with biogenic activities as in hard tissues of fish otoliths. It is supposed to be an important precursor in several calcium carbonate forming processes. Therefore, the studies of vaterite nucleation, growth, and stabilization offer an opportunity to understand the mechanisms of biomineralization and also the processes of crystal nucleation and growth in general. In this work, poly-L-aspartic (pAsp) and poly-L-glutamic (pGlu) acids were chosen as simple analogues to the natural acidic proteins. In order to study the specific polyamino acid/mineral surface interactions, the kinetics of vaterite seed crystal growth was determined in the precipitation systems containing different concentrations of polyamino acids. The experiments were initiated by pouring CaCl2 solution into the same volume of Na2CO3 solution containing vaterite seed crystals. An appropriate polyamino acid concentration was also added to the carbonate component. All experiments were carried out at 25 °C. The propagation of the precipitation process was continuously followed by measuring the pH of the solution. Obtained precipitates were analysed by means of PXRD, FT-IR and SEM. Different theoretical models (parabolic and exponential rate law) were tested and indicated that at given conditions vaterite growth in the model system (system without any additives) and systems with the lower concentrations of additives (c(pAsp or pGlu) < 0.55 ppm) were controlled by the addition of constituting ions into the spiral step emerging from the surface dislocation. Addition of pAsp and pGlu also caused the growth rate inhibition, but at the highest concentrations of used polyamino acids (c(pAsp or pGlu) > 0.7 ppm) the rate controlling mechanism changed as well: it changed from the growth controlled by integration of growth units into the spiral step (parabolic law) to the surface nucleation controlled growth (exponential law).

Calcium carbonate is among the most widely occurring natural minerals. Biogenic calcium carbonate minerals occur in e.g. soils and calcifying marine plankton and are a key component of the global carbon cycle. Abiotic calcium carbonate (synthetic) has a wide range of uses: as a component of pharmaceuticals and foodstuffs, in water treatment, and as a filler in ceramics, plastics, coatings, and paper. Calcium carbonate with desired chemical and structural composition, size and morphology could be tuned by a strict control of precipitation conditions. Amorphous calcium carbonate (ACC), biogenic or synthetic, is an important (meta)stable phase or transient precursor to crystalline calcium carbonate minerals. Depending on the preparation method or its biogenic source, ACC varies in composition, stability and structure. It was found that organisms use organic macromolecules, magnesium, phosphate and structural water to induce ACC formation and to determine its stability. Three model systems with broad range of relative supersaturations, have been investigated previously: (a) Simple system containing only constituent ions: calcium and carbonate, (b) Complex system including constituent ions and their co-ions, sodium and chloride, and (c) Physiological system with the addition of inorganic cation, Mg2+, and at increased ionic strength, 0.15 M NaCl (mimicking the simplified inorganic environment in selected living organisms). In this work, the investigations of ACC stability and transformation were performed for all three model systems. The analyses of precipitation in model systems of different chemical complexity gave the insight into precipitation boundaries and supersaturation domains for formation of different CaCO3 phases. The results have shown that besides the increased initial supersaturation, the complexity of precipitation domain (increased ionic strength, the presence of common inorganic ions, in particular magnesium) has an important role on the formation and properties of the amorphous, metastable and stable CaCO3 phases. The amorphous phases isolated in simple and complex systems have similar properties and transformed to vaterite and cacite. Those formed in physiological system are more stable and transform to monohydrocalcite/aragonite, or remain stable.

Urolithiasis, a form of pathologic biomineralization, is a disease which causes the formation of urinary stones in different parts of kidney or bladder. Recently, increasing number of kidney stones in industrial countries is observed and the interest of scientists to explain their pathogenesis with a special focus on calcium oxalate stones is renewed. Calcium oxalates crystallize in the form of hydrated salts: thermodynamically stable calcium oxalate monohydrate (COM, CaC2O4·H2O, whevellit) and metastable dihydrate (COD, CaC2O4·2H2O, weddellit), and trihydrate (COT, CaC2O4·3H2O). The kidney stones formation under biological conditions can be triggered by various metabolic disorders such as: hipercalciuria, hypocitraturia, hiperoxaluria, and the change in the urine acidity. The mechanisms and the conditions under which they crystallize are still not completely clarified. In this work, the spontaneous precipitation and characterization of calcium oxalate monohydrate under conditions of hiperoxaluria is reported. The experiments were conducted in a simple model system and with the addition of amino acids reportedly important for pathologic biomineralization. The precipitations were carried out with solutions ci(Ca2+) = 7.5 x 10- 3 mol dm-3 and ci(C2O42-) = 6.0 x 10-3 mol dm- 3. The amino acids selected for the addition are often found in the urine of healthy people and in the organic matrix which is an integral part of kidney stones. The reactant solutions were mixed under controlled hydrodynamic and thermodynamic conditions. Changes in the composition and morphology of precipitated calcium oxalate monohydrate were observed by means of PXRD, EPR, SEM, IR and TGA.

Calcium carbonate is main inorganic component of biomineralization in different invertebrate organisms, either in a form of certain polymorph (calcite, vaterite and aragonite) or hydrated modification.The calcite skeletal elements regularly contain small amounts of proteins which are either, incorporated or adsorbed on the single crystals of calcite. Previously it has been shown that isolated fragments of proteins extracted from mineralized tissue, or their synthetic macromolecular analogues, exert a significant influence on the morphology and crystal structure of calcium carbonate when precipitated in the appropriate model systems. In addition, the formation of specific crystal modification can be influenced by parameters like temperature, pH or initial supersaturation, as well as by the presence of inorganic or organic additives. The aim of this research is to investigate distortions of the calcite lattice due to the influence of amino acids, selected as simple models of biomacromolecules supposed to be responsible for nucleation, growth and transformation of calcium carbonates in biomineralizing systems. For that purpose the amino acids having asparagine (Asn), aspartic acid (Asp) and lysine (Lys) were chosen because of differently charged side chains, while the tyrosine (Tyr) and phenylalanine (Phe), as well as serine (Ser) and alanine (Ala) have different polarity. The results of PXRD and EPR techniques as well as growth kinetic data indicate that amino acids interaction with calcite surface increase in the order: Asp > Tyr > Asn > Lys > Ser > Phe > Ala.

A number of recent studies pointed out that the amorphous calcium phosphate (ACP) or amorphous calcium carbonate (ACC) are the first step in either, invertebrate or vertebrate hard tissues formation. Therefore, the renewed interest for in vitro and in vivo investigations of mechanisms of their formation and transformation is not surprising. In spite of the fact that the calcium carbonates and calcium phosphates occur in different organisms, there are a number of similarities in mechanisms of their formation and transformation, as well as in their role in organisms. A number of researchers point out the usefulness of comparing those two systems, but this is still not fully exploited approach. In order to contribute to elucidation of a role of amorphous phases, the physical chemical properties of ACP and ACC formed in the systems at defined and comparable initial experimental conditions (supersaturation, constituent ions ratio, ionic strength and presence of relevant inorganic additives) have been studied. At that, the formation and transformation of ACP and ACC in three model precipitation systems of increased complexity were investigated: (a) system containing only constituent ions, (b) system with increased ionic strength and (c) system containing physiologically relevant co-ions. The experiments were initiated by mixing the reactant solutions at strictly controlled hydrodynamic and thermodynamic conditions, while the progress of reaction was continuously followed potentiometrically. Precipitates obtained after up to 10 minutes were isolated and analysed by means of IC, PXRD, FT-IR, TEM, EPR and TG. The results have shown that ACP is more stable and nucleated at lower relative supersaturations in comparison to ACC formation. Typically, ACC preceded the formation of either, thermodynamically stable calcite or metastable vaterite, aragonite and/or monohydrocalcite, while ACP always transformed to metastable calcium deficiente apatite. The exact transformation mechanisms were found to depend on complexity of the precipitation systems, while the magnesium ions were found to strongly stabilize both amorphous phases.

A need for novel and advanced materials, as well as for their environmentally friendly synthesis is constantly growing. Biomaterials and processes of their formation in organisms (biomineralization) are good pattern for production of such materials. Calcium carbonate (CaCO3), main inorganic component of invertebrate’s hard tissues, is considered to be a relevant biomineralization model. CaCO3 can precipitate (crystallize), either as a polymorph (calcite, vaterite and aragonite), or hydrated and amorphous phase. The calcitic skeletal elements regularly contain small amounts of macromolecules (glycoproteins) which are either, incorporated or adsorbed on the single crystals of calcite. It has been shown previously that the isolated fragments of proteins extracted from mineralised tissue, or their synthetic macromolecular analogues, exert a significant influence on the morphology and crystal structure of calcium carbonate when precipitated in the appropriate model systems. The aim of this research is to investigate a role of the selected amino acids as a simple models of macromolecules supposed to be responsible for precipitation of specific calcium carbonate polymorph or different crystal morphology, during the biomineralization processes. Thus, the aspartic acid (Asp), asparagine (Asn) and lysine (Lys) are selected because of different charge of their side chains, while tyrosine (Tyr) and phenylalanine (Phe), as well as serine (Ser) and alanine (Ala), are chosen because of different polarity. The hypothesis is that not only the acidic, but also the hydrogen bonding amino acid can specifically interact with selected CaCO3 surfaces. Therefore, the Langmuir adsorption constants are calculated from growth kinetic data and used as an indication of extent of organic/inorganic interaction.

Calcite plays an important role in mineralized tissues of many organisms and is very important component in the natural environment as rocks and sediments. The calcitic skeletal elements of many organisms contain small amounts of proteins, which are incorporated within single crystals of calcite. For all of them the crystal size distribution and morphology of the crystals are of great significance and impurities of both inorganic and organic nature play here an important role. The aim of this study is to obtain initial information on the mechanisms of calcitic biominerals formation, in the simple model systems that could be relevant for the initial stages of mineralization. Several amino acids (Tyr, Asp, Lys) were used as models of soluble part of organic matrix that is supposed to be relevant for formation of specific calcium carbonate polymorph and morphology. In order to study changes of local environment of calcite crystal lattice regarding to incorporation of amino acids in calcite, by EPR spectroscopy, Mn2+ was added to systems as structural probe for EPR analysis, along with other analytical methods and technics (SEM, PXRD, FT-IR). The obtained results may shed light on the understanding of protein interaction with mineral phase.

Kalcijev karbonat u beskralježnjacima nastaje procesom biomineralizacije i to u obliku nekog od polimorfa (vaterit, aragonit i kalcit) ili amorfne faze. Pri tome, na nukleaciju određene faze i njenu morfologiju utječu razni čimbenici taloženja, kao na primjer temperatura, prezasićenost, ali i prisutnost otopljenih i/ili neotopljenih anorganskih i organskih komponenata. Proteini, koji su sastavni dio ekstrapalijarne otopine u kojoj se zbiva biomineralizacija, imaju izuzetno važnu ulogu te je stoga u literaturi opisan niz istraživanja utjecaja proteina i njihovih fragmenata izoliranih iz mineraliziranih tkiva, kao i sintetskih (makro)molekulskih analoga. Navedena istraživanja, iako daju važne informacije o utjecaju pojedinih proteina, nepotpuna su, između ostaloga i zbog nerazjašnjenih mehanizama interakcije pojedinih aminokiselina i anorganske faze. – Cilj opisanih istraživanja bio je proučiti utjecaj odabranih aminokiselina, kao jednostavnih modela biomolekula za koje se pretpostavlja da su odgovorne za nukleaciju, rast i transformaciju kalcijeva karbonata u biomineraliziranim sustavima. Kao modeli su korištene asparaginska kiselina (Asp), asparagin (Asn) i lizin (Lys), odabrane zbog različitog naboja bočnih ogranaka, dok se tirozin (Tyr) i fenilalanin (Phe) te serin (Ser) i alanin (Ala) razlikuju u polarnosti molekule. Na temelju FT-IR, XRD, EPR i SEM analiza, praćen je utjecaj odabranih skupina aminokiselina na polimorfizam, morfologiju i distorziju kristalne rešetke stvorenoga kalcita. Pri tome, najizraženije je djelovanje Asp i Tyr: Asp je promjenila morfologiju kristala kalcita kod kojeg se pojavljuje značajan broj novih, stabilnih, romboedarskih ploha, dok dodatak Tyr uzorkuje nastajanje nestabilnih (0 0 1) ploha.

Calcium carbonate is the main inorganic component of biominerals found in many invertebrate organisms and is present either as a specific polymorph (calcite or aragonite), hydrated or in amorphous form. The calcite skeletal elements regularly contain small amounts of proteins which are either incorporated or adsorbed on the single crystals of calcite. Previously it has been shown that isolated fragments of proteins extracted from mineralised tissue, or their synthetic macromolecular analogues, exert a significant influence on the morphology and crystal structure of calcium carbonate when precipitated in the appropriate model systems. The aim of this research is to investigate a role of the selected amino acids as a simple models of biomacromolecules supposed to be responsible for specific precipitation of calcium carbonates in biomineralising systems. In addition, possible crystallographic distortions of the calcite lattice will be investigated. For that purpose amino acids having distinct chemical and physical properties were selected: asparagine, aspartic acid and lysine were chosen because of differently charged side chains, while tyrosine and phenylalanine, as well as serine and alanine have different polarity. The results of structural (EPR) and kinetic analyses indicated an overall inhibition of calcite growth in the presence of all amino acids. The inhibition is probably caused by a slower transformation of initially formed vaterite into the calcite. Since the selected amino acids are charged under the applied experimental conditions, some surface interactions are assumed to be responsible for the observed effect.

Many classes of organic, inorganic or hybrid materials are produced by process of precipitation (crystallization) in which the size, morphology, chemical and structural composition could be tuned by the strict control of formation conditions. In fact, the synthesis of materials that resemble complex properties of natural biominerals is one of key fields in biomimetic science. Calcium carbonates (CaCO3) and calcium phosphates (Ca- PO4) are optimal selection for precipitation model system in biomineralization, because calcium carbonates are the most abundant biominerals that appear in invertebrates, like mollusks, sponges, corals, crustaceans. On the other hand, calcium phosphates (hydroxyapatites) are mostly the constituents of the vertebrate bones and teeth. Calcium carbonates and calcium phosphates appear in a variety of solid phases such as amorphous or crystalline, metastable and stable. The role of the amorphous phase is not completely resolved. The goal of this research is to develop precipitation diagrams of calcium carbonate and calcium phosphate in the concentration relevant for biomineralization of these salts and make their comparison. The precipitated diagrams are performed through screening precipitation experiments in model systems with increased complexity (from basic to physiological systems). Solutions of reagents are mixed together in strictly controlled hydrodynamic and thermodynamic conditions. Precipitation processes are continuously monitored by measuring the pH over time. The resulting solids are separated by filtration at the end of each experiment (1 h, 25 ºC) and analyzed by advanced physical-chemical characterization (XRD, FT-IR, SEM, TEM, EPR, TG). The results of this research give detailed inset to precipitation boundaries and domains in which different solid phases of CaCO3 and Ca-PO4 are precipitated. The formation of amorphous/metastable phases in both carbonate and phosphate system is predominantly influenced by increasing complexity of model systems.

Processes of calcium phosphates (CaP) and calcium carbonates (CaCO3) formation and transformation, due to their biological relevance, are of special interest in the biomimetic preparation of hard tissue implant materials. Both these classes of minerals appear in a number of different solid phases, amorphous or crystalline, stable or metastable, which opens a possibility of fine tuning chemical and structural composition of a biomaterial by a strict control of formation conditions. Although much is known about these systems, the mechanisms of their formation are not yet fully elucidated and their comparison is scarce. The aim of this study is to contribute to the general understanding of mechanisms underlying formation of biologically relevant minerals by comparing properties of solid phases formed in CaP and CaCO3 systems at identical initial experimental conditions (supersaturation, constituent ions ratio, ionic strength and presence of relevant inorganic additives). Three model systems of increased complexity, which contain: (a) only constituent ions, (b) constituent ions and increased ionic strength and (c) constituent ions and physiologically relevant co-ions, were investigated for each class of biominerals. Experiments were initiated by mixing the reactant solutions at strictly controlled hydrodynamic and thermodynamic conditions, while the progress of reaction was continuously followed potentiometrically. Precipitates obtained after 1 hour reaction time were filtrated and analysed by IC, PXRD, FT-IR, TEM, EPR and TG. The results gave insight into the precipitation boundaries and supersaturation domains for formation of different CaP and CaCO3 solid phases. Recent studies have shown that the biominerals in vivo can form via amorphous precursor and its subsequent transformation. Therefore, in each system special attention was given to the formation of amorphous phases. It was found that their transformation into more stable modifications is prolonged by increasing complexity of precipitation system.

Međupovršine kristala biomineralizirajućih soli kalcijevih karbonata i fosfata u dodiru s vodenom otopinom predstavljaju izrazito složeni heterogeni sustav u kojem je prisutan velik broj otopljenih ionskih vrsta i površinskih kompleksa. Pri tome, obje soli iskazuju izuzetan kemijski afinitet spram otopljenih organskih molekula koje sadrže polarne funkcionalne grupe, karboksilnu ili hidroksilnu. U procesu biomineralizacije proteini su glavna organska komponenta ekstracelularne otopine u kojoj se zbiva kristalizacija. Uobičajene strategije istraživanja utjecaja proteina na nastajanje kalcijevih fosfata i karbonata temelje se, bilo na korištenju izoliranih proteina iz mineraliziranog tkiva ili na korištenju njihovih sintetskih analoga, koji po aminokiselinskom sastavu i sekundarnoj strukturi nalikuje prirodnom materijalu. Takva su istraživanja, iako daju važne informacije o utjecaju pojedinog proteina, nepotpuna upravo zbog nepoznate uloge pojedinih aminokiselina.1, 2 Cilj ovog rada bio je istražiti utjecaj tri strukturno različite aminokiseline (alanin (Ala), lizin (Lys) i asparaginska kiselina (Asp)) na kinetiku taloženja te sastav nastale čvrste faze karbonatnih i fostatnih soli. Pri fiziološkim uvjetima Ala je neutralan, Lys je pozitivno, a Asp je negativno nabijen. Istraživanja su provedena pri biološki relevantnim uvjetima (početna prezasićenost, pH, temperatura, ionska jakost) odgovarajućim za kalcijeve karbonate te fosfate i uz različiti dodatak odabrane aminokiseline. Kinetike taloženja kontinuirano su praćene mjerenjem promjene sastava otopine tijekom vremena, a nastala čvrsta faza je karakterizirana pomoću IR spektroskopije te svjetlosne mikroskopije. Istraživanja su pokazala da u sustavima kalcijevih fosfata i kalcijevih karbonata dodatak Asp u ispitivanom koncentracijskom području uzrokuje značajne promjene, dok je djelovanje Lys i Ala znatno manje izraženo.

Istraživanje uvjeta nastajanja kalcijevih karbonata i kalcijevih fosfata važno je prvenstveno zbog njihove uloge u procesu biomineralizacije, no nije zanemariva ni njihova uloga u geologiji, oceanologiji, ekologiji te u različitim industrijskim procesima. Iako su ta dva biominerala različita po svojoj biološkoj ulozi (karbonati su glavna anorganska komponenta mineraliziranih tkiva beskralježnjaka, a fosfati kralježnjaka), procesi njihova nastajanja i transformacije "in vivo", ali i "in vitro" podliježu sličnim pravilima i iskazuju sličan obrazac. Naime, kalcijevi karbonati i kalcijevi fosfati pojavljuju se u nizu različitih čvrtih faza, amorfnih ili kristalnih, metastabilnih i stabilnih, pri čemu je uloga nestabilnih modifikacija djelomično nerasvijetljena.1, 2 Cilj je ovih istraživanja istražiti početne uvjete taloženja kalcijevih karbonata i kalcijevih fosfata u području koncentracija relevantnih za biomineralizaciju tih soli u tipičnim biološkim sustavima te kritički usporediti tako dobivene podatke. Taloženje kalcijevih karbonata i kalcijevih fosfata rađeno je u sustavima u kojima su otopine reaktanata miješane pri strogo kontroliranim hidrodinamičkim i termodinamičkim uvjetima (početna prezasićenost, pH, temperatura, ionska jakost). Proces je kontinuirano praćen mjerenjem koncentracije tijekom vremena, a čvrsta faza je odijeljena filtriranjem. Talog je analiziran IR spektroskopijom te svjetlosnom mikroskopijom. U taložnim dijagramima su određene granice taloženja te koncentracijska područja u kojima nastaju pojedine modifikacije kalcijevih karbonata, odnosno fosfata. Za sustav kalcijevih fosfata je utvrđeno da u cijelom koncentracijskom području, nakon jednog sata, transformacijom primarno nastalog amorfnog kalcijevog fosfata, nastaju kalcijev hidrogenfosfat dihidrat, oktakalcijev fosfat i/ili apatiti. Pri sličnim uvjetima prezasićenosti, u sustavima kalcijevih karbonata nastaje pretežito vaterit, dok je samo u uskom području taložnog dijagrama nastala smjesa vaterita i kalcita.

Calcium carbonates could be biomineralized by different invertebrate organisms, either in a form of certain polymorph (calcite, vaterite and aragonite) or hydrated modification. The formation of specific crystal modification or appearance of appropriate morphological form can be influenced by parameters like temperature, pH or supersaturation, as well as by the presence of inorganic components or biomolecules. At that, the important organic components of extrapallial fluids, in which the biomineralization takes place, are proteins. Previously, the influence of isolated fragments of proteins extracted from mineralized tissues, or their synthetic macromolecular analogues has been investigate in the appropriate calcium carbonate model systems.1, 2 , 3 The aim of this research is to investigate the influence of amino acids, selected as simple models of biomacromolecules supposed to be responsible for nucleation, growth and transformation of calcium carbonates in biomineralizating systems. For that purposes, alanine (Ala), lysine (Lys) and aspartic acid (Asp) were used. Besides their chemical differences, at the conditions applied in the selected model systems the used amino acids have different net charges as well: Ala is neutral, Lys is positive and Asp is negatively charged. The results of potentiometric, spectroscopic, chemical, structural (PXRD) and microscopical (SEM) analyses indicated the overall inhibition of calcite precipitation in the presence of Lys and Aps, that (inhibition) is probably caused by slower transformation of initially formed vaterite, into calcite.4 Since both amino acids are charged at applied experimental conditions, some non specific surface interactions are assumed to be responsible for observed effect. The additional kinetic experiments and molecular modelling will be necessary to reveal the nature of respective intermolecular interactions.

This paper reports on the precipitation of CaCO3 polymorphs, having various crystal morphologies under different conditions. In particular, systems that were subject to ultrasonic irradiation were compared to the corresponding reference systems in the absence of such a treatment. The application of ultrasonic irradiation predominantly resulted in a change of particle size distribution and polymorphic composition of the precipitate, in comparison to the reference systems. Thus, it was found that the supersaturation and temperature influenced the size distribution, in both the reference and sonicated systems. A mixture of calcite, vaterite and aragonite was obtained in all reference systems, at 25 °C. At this temperature, the sonication caused the vaterite content to increase, while aragonite was not detected. In reference and sonicated systems at 80 °C, only aragonite precipitated. The results also indicate that the principle parameter responsible for the morphology of vaterite was the initial supersaturation: at higher supersaturation spherical vaterite particles precipitated, while at lower supersaturation hexagonal platelets were obtained. The morphological investigations also indicated different mechanisms of vaterite formation in the systems in which precipitation was initiated at higher supersaturation: spherulitic growth of vaterite was observed in sonicated systems, while the aggregation of primary particles was predominant in the reference systems. At lower supersaturation, the effect of c(Ca2+)/c(CO32-) on the morphology of hexagonal platelets of vaterite was observed as well. By varying the c(Ca2+)/c(CO32-), significant changes of the polymorphic composition were observed only in the sonicated systems, at 25 ˚C.

A method for preparation of significant amount of hollow rhombohedral calcite nanoparticles, based on carbonation of calcium hydroxide suspension, is described. The mineralogical and morphological analyses of the precipitate confirmed the existence of exclusively stable polymorphic modification, calcite, with the mean particle size of about 100 nm and the diameter of the holes observed at the surfaces that are about 50 nm. The analysis of carbonation kinetics pointed out to a complex mechanism of hollow particles formation at high initial supersaturation, that assumed nucleation of amorphous precursor calcium carbonate phase and its solution mediated transformation into nanosized crystalline calcite. The holes obtained at the calcite surfaces are most probably the imprints remained after the dissolution of amorphous calcium carbonate particles.

Mnogi živi organizmi, napose životinjski, formiraju mineralne strukture, biološki kontroliranim procesom poznatim kao biomineralizacija. Tako stvoreni materijali su prirodni organsko-anorganski kompoziti, pri čemu je neki od polimorfa kalcijeva karbonata jedna od češćih mineralnih komponenti. Tako se npr., ljuštura morskog puža Haliotis rufescens sastoji od kalcitnih i aragonitnih slojeva te u njih ugrađenih karakterističnih biopolimernih molekula za koje se pretpostavlja da su odgovorne za biomineralizaciju. U ovom je radu, istraženo djelovanje intersticijskog polipeptida izoliranog iz zelenog sloja ljušture morskog puža, GP, na proces spontanog taloženja polimorfa kalcijeva karbonata iz prezasićenih vodenih otopina. Pri tome su proučavana tri modelna sustava, različitih početnih prezasićenosti, u kojima je, posljedično, i početni mineraloški sastav taloga bio različit. U sustavu najniže prezasićenosti taložio je kalcit, dok je u sustavu više prezasićenosti inicijalno taložila smjesa kalcita i vaterita. U sustavu koji je po sastavu glavnih anorganskih komponenti podražavao plaštenu tekućinu (morsku vodu), taložio je isključivo aragonit. Analizom kinetičkih podataka procesa taloženja te rentgenskom analizom, FT-IR spektroskopijom, svjetlosnom konfokalnom mikroskopijom i pretražnom elektronskom mikroskopijom je utvrđeno da dodatak GP, u proučavanim sustavima uzrokuje inhibiciju taloženja i promjenu morfologije kristala, što je posljedica, kako adsorpcije GP na plohe kristala, tako i njegove ugradnje u strukturu mineralne matrice.

One of the most abundant minerals in nature and consequently one of the most intensively investigated precipitation system is calcium carbonate, CaCO3. Calcium carbonate forms six solid modifications: three polymorphs (calcite, aragonite and vaterite), calcium carbonate hexahydrate, calcium carbonate monohydrate and amorphous calcium carbonate. In biological systems, CaCO3 predominantly appears as calcite and/or aragonite and less commonly as vaterite and/or amorphous calcium carbonate. Calcium carbonate biominerals (formed during the biomineralization) are natural biocomposites in which the inorganic phase is principal component with the organic matrix being present as a minor fraction.1 Molluscs shells, pearls and corals are examples of such natural biocomposites. Extraordinary physical and chemical properties of these materials are the result of a controlled crystallization of CaCO3, that is, essentially, induced by the organic matrix. Aragonite, one of calcium carbonate polymorphs, is the major mineral component in corals. Due to the considerable susceptibility of corals on temperature changes, the global warming is one of the greatest threats for corals and coral reefs existence. In addition the increased atmospheric CO2 reacts with sea water and causes its acidification, which may influence the CaCO3 precipitation in calcifying organisms such as corals. An accurate evaluation of global warming effects on corals requires the knowledge of coral biomineralization processes that is still poorly understood In order to determine the role of soluble, hydrophilic, organic macromolecules (SOM), in a process of coral biomineralization, the effects of soluble biomacromolecules extracted from the Mediterranean coral skeleton of Balanophyllia europea2 and Leptosammia pruvoti, on calcium carbonate precipitation has been investigated. The results of kinetics and structural analysis indicated that the respective macromolecules of SOM strongly interact with aragonite crystals. References: 1 Mann, S., (2001). Biomineralization. Principles and Concepts in Bioinorganic Materials Chemistry, Oxford University Press, New York. 2 Goffredo, S., Vergni, P., Reggi, M., Caroselli, E., Sparla F., Levy O., Dubinsky Z.  Falini, G. (2011). PLoS ONE 6(7) e22338.

Many organisms exhibit highly specific control of mineralization by using biomolecules [1]. Molluscs shells, pearls and corals are examples of natural biocomposites in which calcium carbonate (CaCO3) is the principal mineral component. Mechanical and chemical properties of these materials are the results of a controlled crystallization of CaCO3 crystals, that is, essentially, induced by the organic matrix containing proteins reach in acidic amino acid residues. In this work, the effect of a water soluble polypeptide, extracted from green layer of the abalone shell (GP), on the process of spontaneous CaCO3 precipitation was investigated. Although the green sheet is reported to be unique to the abalone shell, it represents a good model for studies of molluscan shell biomineralization, because of observed nucleating activity [2]. Since GP has composition and structure similar to those of the parent green sheet, it can be used as model in nucleation experiments. As the CaCO3 precipitation models, three systems were investigated. Thus, system (1) imitated the sea water composition and only aragonite spontaneously precipitated. System (2) is of lower initial supersaturation and only calcite precipitated in the absence of GP, while the initial supersaturation in system (3) is relatively high and mixture of calcite and vaterite initially precipitated. In systems (2) and (3) the precipitation was initiated by mixing equimolar concentrations of Na2CO3 and CaCl2. All experiments were conducted at t = 21 °C and the progress of precipitation was followed by recording pH as a function of time. Samples were characterized by means of FT-IR spectroscopy, X-ray diffraction and optical and scanning electron microscopy. It was found that GP inhibited precipitation in all model systems, while the significant morphological changes were observed on calcite crystals only. It could be concluded that adsorption of GP onto the surface of the growing crystals is responsible for the inhibition effects.

Biomineralizacijom ljuštura nekih školjaka nastaju prirodni biokompozitni materijali izuzetnih mehaničkih svojstava. Ovi materijali su, zapravo, anorgansko-organske kompozitne tvorevine. Anorgansku komponentu čini kalcijev karbonat (CaCO3). Maseni udio organske komponente može biti vrlo malen, npr. svega 5% u oklopima školjaka, međutim njena funkcija je izuzetna jer služi kao templat u formiranju ovakvih biomaterijala. Utvrđeno je da u procesu biomineralizacije oklopa školjaka ključnu ulogu imaju kiseli glikoproteini, bogati asparaginskom (Asp) i glutaminskom (Glu) kiselinom. Kako bi u laboratorijskim uvjetima proučili mehanizam nastajanja takvih izuzetnih biokompozitnih materijala, rađeni su pokusi taloženja iniciranog dodatkom kristalnoga sjemena polimorfa kalcijeva karbonata, kalcita, uz dodatak poli-L-asparaginske kiseline (pAsp), poli-L-glutaminske kiseline (pGlu) ili poli-L-lizina (pLys). Pri tom su pAsp i pGlu podražavali ulogu kiselih, prirodnih, glikoproteina u procesu biomineralizacije. Analizom izračunatih kinetičkih parametara pronađeno je da se rast kalcita zbiva na spiralnoj dislokaciji te da je integracija konstitucijskih iona u kristalnu rešetku najsporiji proces. Dodatak pAsp ili pGlu uzrokuje inhibiciju rasta kristala, no također i promjenu mehanizma, tako da je rast kontroliran površinskom nukleacijom. Efekt je najizraženiji u sustavu kalcit / pAsp, što se može pripisati specifičnoj koordinativnoj vezi između karboksilnih skupina bočnih ogranaka pAsp, pretežno planarno uređene strukture (β-nabrani list), i Ca2+ iona s površine kalcita.

Biomineralizacijom oklopa školjaka nastaju prirodni biokompozitni materijali izuzetnih mehaničkih i kemijskih svojstava, u kojima mineralnu komponentu čine neki od polimorfa kalcijeva karbonata (CaCO3). Tijekom tog procesa ključnu ulogu imaju kiseli glikoproteini bogati glutaminskom (Glu) i asparaginskom kiselinom (Asp). S nakanom rasvjetljavanja uloge prirodnih polipeptida tijekom nastanka tih izuzetnih biokompozitnih materijala provedeni su pokusi u kojima je istraženo djelovanje poli-L-asparaginske kiseline (pAsp), poli-L-glutaminske kiseline (pGlu) i poli-L-lizina (pLys) na spontano taloženje CaCO3. Pri tome, pAsp i pGlu podražavaju ulogu prirodnih, kiselih, proteina u procesima biomineralizacije oklopa školjaka dok pLys služi kao kontrola. Kao modelni sustavi odabrani su sustav niže početne prezasićenosti (1), u kojem je spontano taložio isključivo vaterit te sustav više početne prezasićenosti (2) u kojem je spontano taložila smjesa kalcita i vaterita. Dodatak pGlu i pAsp, u sustav (1) uzrokovao je značajnu inhibiciju nukleacije, dok je u sustavu (2) osim inhibicije uzrokovao i promjenu morfologije te sastava taloga. S porastom koncentracije pAsp i pGlu smanjivao se maseni udio kalcita, što je moguće objasniti jačim vezanjem kiselih polipeptida na površinu kalcita nego na površinu vaterita te jačom inhibicijom nukleacije i rasta kristala kalcita. Pri tom se inhibicijsko djelovanje polipeptida smanjivalo u nizu: InhpAsp > InhpGlu >> InhpLys [Ref: Njegić–Džakula, B., Falini, G., Brečević, Lj., Skoko, Ž., Kralj, D., J. Colloid Interface Sci. 343 (2010) 553-563].

Biomineralizacijom ljuštura školjaka nastaju prirodni biokompozitni materijali izuzetnih mehaničkih svojstava, pri čemu je utvrđeno da je u tom procesu ključna uloga kiselih glikoproteina, bogatih glutaminskom (Glu) i asparaginskom kiselinom (Asp). Kako bi u laboratorijskim uvjetima proučili mehanizam nastajanja takvih izuzetnih biokompozitnih materijala, u kojima mineralnu komponentu čine neki od polimorfa kalcijeva karbonata (CaCO3), rađeni su pokusi spontanog taloženja te taloženja iniciranog dodatkom kristalnoga sjemena polimorfa CaCO3, kalcita i vaterita, uz dodatak poli-L-asparaginske kiseline (pAsp), poli-L-glutaminske kiseline (pGlu) ili poli-L-lizina (pLys). Pri tome, pAsp i pGlu podražavaju ulogu kiselih, prirodnih, glikoproteina u procesu biomineralizacije. Kao modelni sustavi odabrani su (a) sustav niže početne prezasićenosti, , u kojem je spontano taložio isključivo vaterit te (b) sustav više početne prezasićenosti, u kojem je spontano taložila smjesa kalcita i vaterita. U sustavima u kojima je proučavano spontano taloženje kalcijeva karbonata, utvrđeno je da dodatak pGlu i pAsp u sustav (a) uzrokuje značajnu inhibiciju nukleacije, dok je u sustavu (b) osim inhibicije, dodatak kiselih polipeptida uzrokovao i promjenu morfologije te sastava taloga. S porastom koncentracije pAsp i pGlu smanjivao se maseni udio kalcita, što je objašnjeno jačim vezanjem kiselih polipeptida na površinu kalcita te jačom inhibicijom nukleacije i rasta kristala kalcita. Kinetički parametri te utvrđeni mehanizmi kristalnoga rasta, ukazuju da se rast kalcita i vaterita zbiva na spiralnoj dislokaciji te da je integracija konstitucijskih iona u kristalnu rešetku najsporiji proces. Dodatak pAsp ili pGlu uzrokuje inhibiciju rasta kristala, no također i promjenu mehanizma, tako da je rast kontroliran površinskom nukleacijom.

Calcium carbonate polymorphs (calcite, aragonite and vaterite) are probably the most common biominerals, at least the two more stable ones (calcite and aragonite). In the case of mollusc shells, regular calcite and/or aragonite mineral layers are associated with organic matrix, thus resulting in hybrid materials having remarkable mechanical properties. Water-soluble components of organic matrix, mostly acidic proteins rich in aspartic and glutamic acid residues, are supposed to be responsible for the formation of a particular polymorph and for arranging its growth into desired patterns by controlling the shape, size and orientation of single crystals. In this work, poly-L-glutamic (pGlu) and poly-L-aspartic acid (pAsp) were chosen as simple analogues to the mentioned soluble acidic proteins. Although both polypeptides have similar chemical composition, they differ in structure when in solution containing calcium ions: pAsp is predominantly in the form of  sheet and pGlu is predominantly in random coil conformation. In order to study the specific polypeptide/mineral surface interactions, the kinetics of calcite seed crystal growth was determined in the precipitation systems containing different concentrations of polypeptides. It was found that, for the whole range of the investigated initial supersaturations, the crystal growth rate of calcite was a parabolic function of supersaturation, indicating that the integration of constituent ions into the spiral step was the rate determining process. Both, pAsp and pGlu inhibited the crystal growth rate of calcite, the effect being more pronounced in the case of pAsp. Moreover, the exponential relationship between the crystal growth rate and supersaturation was found when either pGlu or pAsp was present in the solution. This indicated that the surface nucleation was the controlling mechanism. The obtained results suggest the possible role of the polypeptide structure on the extent and the type of their interactions with the calcite surface.

Slojeviti dvostruki hidroksidi čine veliku skupinu prirodnih i sintetičkih minerala čija se struktura izvodi iz strukture brucita - magnezijeva hidroksida, Mg(OH)_2. Kod tih spojeva, poznatih i kao anionske gline, Mg^2+ je smješten u oktaedarskom sloju i djelomično je zamijenjen nekim trovalentnim ionom slične veličine. Stvoreni višak pozitivnog naboja kompenziran je interkalacijom odgovarajućih aniona, najčešće nitrata, klorida ili karbonata, u međuslojeve, a kako se radi o pretežito elektrostatskim silama njihova izmjena s drugim negativno nabijenim ionima je lako moguća. Opća formula takvih spojeva je Mg(1-x)M^3+x(OH)_2(Am^-)x/m • nH_2O, gdje je M^3+ trovalentni kation, najčešće Al^3+, a Am^- je izmjenjivi anion. Interes za istraživanje svojstava i metoda priprave ove skupine spojeva vezan je uz njihovu moguću primjenu: mineralni hidroksilni sloj može poslužiti kao zaštita od degradacije velikih kompleksnih i/ili organskih aniona (katalizatori i biološki aktivne tvari – lijekovi, vitamini, DNA), odnosno mogu biti korišteni za kontrolirano otpuštanje aktivnih tvari uslijed procesa ionske izmjene. U ovom su radu prikazani rezultati priprave taloga anionske gline dvjema različitim metodama ; tzv. metodom koprecipitacije i metodom ionske izmjene. Približni sastav taloga moguće je prikazati formulom Mg0, 68Al0, 32(OH)_2A0, 32/m • nH_2O u kojoj n varira od 0, 5 do 1, 2, m je naboj aniona, a A je NO^3-, Cl^- ili C_6H_5O^73-. Svojstva taloga, poglavito veličina čestica te s njome povezana kinetika i doseg ionske izmjene, korelirana su s uvjetima priprave: temperaturom i brzinom titracije reakcijskih komponenti.