The locus of mineral crystallites in bone

The organic content of mineralized tissues has been found to decrease with increasing tissue density, from about 60% of the mineral weight in light bone like deer antler to 1 to 2% in hyperdense bone like porpoise petrosal. The ratio of the weight of mineral that can fill the collagen hole zones to the total mineral content can be no greater than 20% for deer antler and decreases to less than 5% for hyperdense bone. Moreover, the dimensions of hydroxyapatite crystallites have been determined by various investigators to be larger than the intermolecular spacing of collagen molecules. Such crystallites can only be fitted within the collagen fibril if collagen molecules are packed differently from the accepted models. Electron micrographs of fish dentin, at a very early stage of mineralization, show the needle-like crystallites lying in dense strips between collagen fibrils and practically no crystallites within the fibrils. A similar pattern of dense strips of crystallites between fibrils can be identified in examples from more advanced stages of mineralization, taken from fish dentin, cat dentin and cow tibia, even though some of the needle-like crystallites are superimposed on the fibril banded pattern. In every instance there are regions of the fibrils where there are no visible needle-like crystallites. Examination of the work of others shows a similar distribution of the mineral component, except that none exactly resemble the micrograph of the earliest stage of fish dentin provided in this report. The collagen banding is observed to be in spatial phase over many fibrils. The needle-like crystallites may be observed to be bunched in phase with the collagen banding and with the same spatial periodicity. The bunching is most obvious in the least densely mineralized specimens. This observation can account for the x-ray and neutron diffraction patterns which shown the axial period of the mineral to be like that of the collagen axial macroperiod and to be in phase with the hole zones of collagen fibrils. These prior studies were interpreted to show that the crystallites must be within the hole zones. Our images are interpreted to show that most of the mineral is outside of the collagen fibrils in the extrafibrillar volume. The interpretation is in agreement with neutron diffraction studies of various mineralized tissues as well as with earlier diffraction studies of mineralized turkey leg tendon and with the calculations of the amount of mineral that can be contained within the collagen of mineralized tissue.     

Differences between top-down and bottom-up approaches in mineralizing thick, partially demineralized collagen scaffolds

Biominerals exhibit complex hierarchical structures derived from bottom-up self-assembly mechanisms. Type I collagen serves as the building block for mineralized tissues such as bone and dentin. In the present study, 250-300 μm thick, partially demineralized collagen scaffolds exhibiting a gradient of demineralization from the base to surface were mineralized using a classical top-down approach and a non-classical bottom-up approach. The top-down approach involved epitaxial growth over seed crystallites. The bottom-up approach utilized biomimetic analogs of matrix proteins to stabilize amorphous calcium phosphate nanoprecursors and template apatite nucleation and growth within the collagen matrix. Micro-computed tomography and transmission electron microscopy were employed to examine mineral uptake and apatite arrangement within the mineralized collagen matrix. The top-down approach could mineralize only the base of the partially demineralized scaffold, where remnant seed crystallites were abundant. Minimal mineralization was observed along the surface of the scaffold; extrafibrillar mineralization was predominantly observed. Conversely, the entire partially demineralized scaffold, including apatite-depleted collagen fibrils, was mineralized by the bottom-up approach, with evidence of both intrafibrillar and extrafibrillar mineralization. Understanding the different mechanisms involved in these two mineralization approaches is pivotal in adopting the optimum strategy for fabricating novel nanostructured materials in bioengineering research.     

Role of phosphophoryn in dentin mineralization

Mineral deposition is essential for the development of hard tissues like bone and teeth. In matrix-mediated mechanisms responsible for dentin formation, type I collagen defines the framework for mineral deposition and by itself is not sufficient to support nucleation of hydroxyapatite. However, in the presence of non-collagenous proteins, nucleation sites have been identified within the hole regions of the fibrils, and at these sites, mineral crystals can grow and propagate. Non-collagenous proteins constitute 5-10% of the total extracellular matrix proteins. They are embedded within the mineral deposits, suggesting a possible interaction with the mineral phase. During dentin formation, phosphophoryn (PP), an abundant macromolecule in the extracellular matrix, can initiate mineral deposition in localized regions by matrix-mediated mineralization mechanism. In our work, we have demonstrated that PP, due to its highly phosphorylated post-translational modification, can bind calcium ions with high affinity and at the same time aggregate collagen fibrils at the mineralization front. Molecular modeling has further demonstrated that the spacing of the carboxyl and phosphate groups present on PP might be essential for dictating the crystal orientation relative to the collagen substrate. Thus, PP may provide the interface linkage between mineral crystal and collagen fibrils.     

Development of bone-like composites via the polymer-induced liquid-precursor (PILP) process. Part 1: Influence of polymer molecular weight

Bone is an organic–inorganic composite consisting primarily of collagen fibrils and hydroxyapatite crystals intricately interlocked to provide skeletal and metabolic functions. Non-collagenous proteins (NCPs) are also present, and although only a minor component, the NCPs are thought to play an important role in modulating the mineralization process. During secondary bone formation, an interpenetrating structure is created by intrafibrillar mineralization of the collagen matrix. Many researchers have tried to develop bone-like collagen–hydroxyapatite (HA) composites via the conventional crystallization process of nucleation and growth. While those methods have been successful in inducing heterogeneous nucleation of HA on the surface of collagen scaffolds, they have failed to produce a composite with the interpenetrating nanostructured architecture of bone. Our group has shown that intrafibrillar mineralization of type I collagen can be achieved using a polymer-induced liquid-precursor (PILP) process. In this process, acidic polypeptides are included in the mineralization solution to mimic the function of the acidic NCPs, and in vitro studies have found that acidic peptides such as polyaspartate induce a liquid-phase amorphous mineral precursor. Using this PILP process, we have been able to prepare collagen–HA composites with the fundamental nanostructure of bone, wherein HA nanocrystals are embedded within the collagen fibrils. This study shows that through further optimization a very high degree of mineralization can be achieved, with compositions matching that of bone. Synthetic collagen sponges were mineralized with calcium phosphate while analyzing various parameters of the reaction, with the focus of this report on the molecular weight of the polymeric process-directing agent. In order to determine whether intrafibrillar mineralization was achieved, an in-depth characterization of the mineralized composites was performed, including wide-angle X-ray diffraction, electron microscopy and thermogravimetric analyses. The results of this work lead us closer to the development of bone-like collagen–HA composites that could become the next generation of synthetic bone grafts.     

Biomimetic mineralization of collagen fibrils induced by amine-terminated PAMAM dendrimers – PAMAM dendrimers for remineralization

Objective: Achieving biomimetic mineralization of collagen fibrils by mimicking the role of non-collagenous proteins (NCPs) with biomimetic analogs is of great interest in the fields of material science and stomatology. Amine-terminated PAMAM dendrimer (PAMAM-NH₂), which possesses a highly ordered architecture and many calcium coordination sites, may be a desirable template for simulating NCPs to induce mineralization of collagen fibrils. In this study, we focused on the ability of PAMAM-NH₂ to mineralize collagen fibrils. Design: Type-I collagen fibrils were reconstituted over 400-mesh formvar-and-carbon-coated gold grids and treated with a third-generation PAMAM-NH₂ (G3-PAMAM-NH₂) solution. The treated collagen fibrils were immersed in artificial saliva for different lengths of time. The morphologies of the mineralized reconstituted type-I collagen fibrils were characterized by transmission electron microscopy. Results: No obvious mineralized collagen fibrils were detected in the control group. On the contrary, collagen fibrils were heavily mineralized in the experimental group. Most importantly, intrafibrillar mineralization was achieved within the reconstituted type-I collagen fibrils. Conclusions: In this study, we successfully induced biomimetic mineralization within type-I collagen fibrils using G3-PAMAM-NH₂. This strategy may serve as a potential therapeutic technique for restoring completely demineralized collagenous mineralized tissues.     

力学环境调控骨基质仿生矿化

骨缺损一直以来都是威胁人类生命健康的重要原因,人工仿生骨修复替代材料是目前治疗骨损伤最为有效、 可行的解决途径之一。 要研发人工骨仿生材料,必先构建体外仿生矿化体系,以研究天然骨基质的矿化机制。 胶 原是矿化发生的模板,其交联度、直径、渗透压和表面电荷等性质会直接影响矿化的进行。 矿化发生的生化和力学 环境对矿化过程的影响也十分明显,特别是非胶原蛋白和流体切应力。 流体切应力是骨组织在微观环境下受到的 最主要力学刺激方式,对骨骼生长、修复以及健康维护都具有重要意义。 不同水平和加载方式的切应力对无定形 磷酸钙向骨磷灰石的转化、胶原纤维的自组装和定向排列以及分层纤维内矿化的形成具有显著作用。 本文总结影 响骨基质矿化的因素及其作用机制,重点介绍流体切应力对胶原矿化的调控作用,并展望未来的发展方向。     

Bone regeneration by using scaffold based on mineralized recombinant collagen

Bone regeneration was achieved in the 15-mm segmental defect model in the radius of rabbit by using the scaffold based on mineralized recombinant collagen for the first time. The recombinant collagen was recombinant human-like type I collagen, which was produced by cloning a partial cDNA that was reversed by mRNA from human collagen alpha1(I) and transferred to E. coli. The scaffold material nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA) was developed by biomimetic synthesis. Thermo gravimetric analysis, X-ray diffraction and scanning electron microscopy were applied to exhibit that the scaffold showed some features of natural bone both in main component and hierarchical microstructure. The percentages of organic phase and inorganic phase of nHA/RHLC were similar to that of natural bone. The three-dimensional porous scaffold materials mimic the microstructure of cancellous bone. In the implantation experiment, the segmental defect was healed 24 weeks after surgery, and the implanted composite was completely substituted by new bone tissue. The results of the implantation experiment were very comparable with that of the scaffold based on mineralized animal-sourced collagen. It is concluded that the scaffold based on mineralized recombinant collagen maintains the advantages of mineralized animal-sourced collagen, while avoids potential virus-dangers. The scaffold is a promising material for bone tissue engineering.     

Dentin matrix proteins: composition and possible functions in calcification

Dentin may be regarded as a mineralized connective tissue. In its composition as well as its mode of formation, dentin exhibits several similarities with bone, but also definite differences. The dentin organic phase, the matrix, determines its morphology and is believed to be instrumental in the formation of the mineral phase. A fibrous web of collagen type I dominates the organic matrix. Also, minor amounts of other collagen types may be present. The noncollagenous proteins (NCPs), which constitute about 10% of the matrix, fall into several categories: phosphoproteins, Gla-proteins of the osteocalcin type as well as matrix Gla-protein, proteoglycans, different acidic glycoproteins, and serum proteins. Some of these NCPs have unique chemical compositions that give them specific properties. Dentinogenesis occurs by two simultaneous processes: the formation of a collagenous web in predentin, which is followed by the formation of the inorganic phase at the mineralization front. The composition of the predentin organic matrix differs from that of dentin, as some NCP components are secreted extracellularly just in advance of the mineralization front. In addition, some constituents of predentin seem to be metabolized. The NCPs may be important to several processes during dentinogenesis. Much evidence indicates that noncollagenous components in the matrix are instrumental in mineral formation. New data show that polyanionic NCPs, such as phosphoprotein and proteoglycans, when immobilized on a solid support, induce apatite formation under physiological conditions. These data indicate that polyanionic NCPs may function as mineral nucleators in vivo. They may also act as size and rate regulators for crystallization and promote calcium ion diffusion in the tissue. In addition, NCPs may regulate collagen fibrillogenesis.     

Considerations regarding the structure of the mammalian mineralized osteoid from viewpoint of the generalized packing model

The relative magnitudes of mineral, organic and water contents of dense mammalian bone are calculated by a new theory based on recent findings: (1) The neutron diffraction studies of mineralized tissues with different densities demonstrated an inverse relationship between wet density and the equatorial diffraction spacing of the collagen. (2) The neutron studies showed there was very little mineral within the collagen fibrils. (3) A generalized packing model for collagen has been advanced to show how the equatorial spacing can be varied depending on tissue type, water content, and mineral content. (4) The water content of collagen fibrils when calculated from the generalized packing model matches the experimentally determined values for rat tail tendon fibers, bone matrix, and fully mineralized bone. A computational model was developed based on the generalized packing model. It provides a unifying approach to explain many features of mineralized fibrous collagenous tissues. The results are presented as estimates of the mineralized collagen fibril density, the volume fraction of collagen in bone, the density of the extrafibrillar space, the fraction of the e.f. space occupied by mineral and the ratio of mineral within collagen to total mineral content, each expressed as a function of wet bone density. A useful data base, available from previous studies, related mineral, organic and water weight fractions to wet bone density, for a density range from 1.7 g/cc for deer antler to 2.7 g/cc for porpoise petrosal. A second order polynomial was found for each weight fraction component, with bone density as the input variable, with a standard deviation less than 2% of total bone weight. This permits the bone properties to be related to a single variable, the wet bone density. It is seen that compacting the collagen fibrils as well as reducing the organic component weight fraction are two important factors determining the structure of the mineralized osteoid. It was concluded that voids and pore spaces may occupy at least 5% of the bone volume.     

Functional biomimetic analogs help remineralize apatite-depleted demineralized resin-infiltrated dentin via a bottom–up approach

Natural biominerals are formed through metastable amorphous precursor phases via a bottom–up, nanoparticle-mediated mineralization mechanism. Using an acid-etched human dentin model to create a layer of completely demineralized collagen matrix, a bio-inspired mineralization scheme has been developed based on the use of dual biomimetic analogs. These analogs help to sequester fluidic amorphous calcium phosphate nanoprecursors and function as templates for guiding homogeneous apatite nucleation within the collagen fibrils. By adopting this scheme for remineralizing adhesive resin-bonded, completely demineralized dentin, we have been able to redeposit intrafibrillar and extrafibrillar apatites in completely demineralized collagen matrices that are imperfectly infiltrated by resins. This study utilizes a spectrum of completely and partially demineralized dentin collagen matrices to further validate the necessity for using a biomimetic analog-containing medium for remineralizing resin-infiltrated partially demineralized collagen matrices in which remnant seed crystallites are present. In control specimens in which biomimetic analogs are absent from the remineralization medium, remineralization could only be seen in partially demineralized collagen matrices, probably by epitaxial growth via a top–down crystallization approach. Conversely, in the presence of biomimetic analogs in the remineralization medium, intrafibrillar remineralization of completely demineralized collagen matrices via a bottom–up crystallization mechanism can additionally be identified. The latter is characterized by the transition of intrafibrillar minerals from an inchoate state of continuously braided microfibrillar electron-dense amorphous strands to discrete nanocrystals, and ultimately into larger crystalline platelets within the collagen fibrils. Biomimetic remineralization via dual biomimetic analogs has the potential to be translated into a functional delivery system for salvaging failing resin–dentin bonds.     

Preparation of bone-like apatite-collagen nanocomposites by a biomimetic process with phosphorylated collagen

By imitating in vivo bone mineralization, bone-like apatite-collagen nanocomposites were prepared by chemical phosphorylation of collagen and subsequent biomimetic growth of bone-like nanoapatite on collagen nanofibers. Two steps were employed in the composites preparation. First, the collagen was phosphorylated by chemical treatment, which provides the nucleation sites for bone-like apatite mineralization. The subsequent growth of bone-like nanoapatite on the phosphorylated collagen nanofibers was performed in simulated body fluid (SBF). The characterization of the composites showed that the composites were composed of nanoapatite mineralized collagen nanofibers that exhibit similarity to natural bone in composition and crystal morphology.     

Dentin Matrix Proteins and Dentinogenesis

The precise mechanisms involved in dentinogenesis are not understood; however, the information to date suggests that a number of highly controlled extracellular events are involved. Mature odontoblasts secrete collagen at the cell border into predentin. They synthesize and secrete other non-collagenous proteins (NCPs) at the mineralization front, possibly through odontoblastic processes. A collagen-NCP complex is formed at the predentin-dentin border and apatite crystal initiation and growth takes place. One of the research needs is to uncover the nature of this dentin collagen-NCP complex and to understand how it controls mineralization. At least three dentin specific NCPs are known: phosphophoryn(s), dentin sialoprotein (DSP) and AG1 (Dmp1). Other macromolecules are commonly made by osteoblasts and odontoblasts and participate in bone and dentin formation.

Some progress in understanding dentin mineralization has been gained by focusing upon the role of phosphophoryns. These highly phosphorylated proteins are secreted at the mineralization front, where a small portion binds in the gap region of type I collagen fibrils. This portion of phosphoproteins probably initiates formation of plate-like apatite crystals. Additional phosphophoryns in higher concentrations bind to the growing apatite crystals and slow their growth, possibly influencing their size and shape.

Other areas which need careful investigations are those involving the mechanisms involved in odontoblast differentiation, how the synthesis of the dentin specific NCPs is controlled and the precise roles of these macromolecules in dentinogenesis. Future experimentation will focus on the gene structures for these NCPs and the mechanisms of tissue specific gene regulation. Tests for function can then be pursued in “gene knockout” experiments. There is no doubt that current “new” scientific approaches being utilized to answer many scientific questions in other fields will greatly impact our ability to answer the questions surrounding the process of dentinogenesis.     

Dentin Mineralization and the Role of Odontoblasts in Calcium Transport

Dentin is formed by two simultaneous processes, in which the odontoblasts are instrumental—the formation of the collagenous matrix, and mineral crystal formation in this matrix. This pattern of formation is similar to that of bone, another mineralized connective tissue. Dentin and bone also have chemical compositions which are similar but with distinct differences. It is of fundamental importance to understand how the ions constituting the inorganic phase are transported from the circulation to the site of mineral formation and how this transport is regulated. For dentinogenesis, calcium is essentially the only ion for which data are available. Recent evidence suggests that a major portion of the Ca²⁺ ions are transported by a transcellular route, thus being under cellular control. The cells maintain a delicate Ca²⁺ ion balance by the concerted action of transmembraneous transport mechanisms, including Ca-ATPase, Na⁺/Ca²⁺ exchangers and calcium channels, and of intracellular Ca²⁺-binding proteins. The net effect of this is a maintenance of a sub-micromolar intracellular Ca²⁺ activity, and an extracellular accumulation of Ca²⁺ ions in predentin, at the mineralization front. Predentin can be regarded as a zone of formation and maturation of the scaffolding collagen web of the dentin organic matrix. In addition to collagen, it contains little but proteoglycan. Simultaneous with mineral formation, additional non-collagenous macromolecules are added to the extracellular matrix of dentin, these presumably being transported within the odontoblast process. Among these are highly phosphorylated dentin phosphoprotein (phospho-phoryn) and another pool of proteoglycan. The functionality of this may be explained by the fact that polyanionic macromolecules are capable of inducing the formation of hydroxyapatite at ionic conditions resembling those in vivo. They can also inhibit mineral growth and regulate crystal size.     

The preparation and characteristics of a carbonated hydroxyapatite/collagen composite at room temperature

Nanocarbonated hydroxyapatite/collagen (nCHAC) composite was prepared at room temperature via biomimetic self-assembly method. X-ray diffraction (XRD), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) were performed. This composite shows the same inorganic phase of natural bone with nanosized level and low degree of crystallinity, and contains 2.8-14.7 wt % of carbonated content. TEM results confirm that the microstructure of this composite is the mineralized collagen fiber bundle like the hierarchical structure of natural bone. The diameter of a single mineralized collagen fiber is about 4 nm. Slightly different assembly units of the composite with different carbonates and collagen were demonstrated. The carbonated percentage affects the mineral crystal size and collagen fibril assembly. Because of the biomimetic component and microstructure, the use of nCHAC composite is promising for hard tissue therapy.     

The three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium-phosphate crystals of pickerel and herring fish bone

High-voltage (1.0 mega-volt) electron stereomicroscopy has been used to examine the spatial relationship between the inorganic crystals and the collagen fibrils of pickerel and herring bone. Stereomicrographs of cross sections of the collagen fibrils encompassing regions of initial to full mineralization showed that the calcium phosphate crystals are located within the collagen fibrils. In all stages of mineralization, calcium-phosphate deposits were not observed associated or within membrane-bound structures. Serial cross sections of the fully mineralized collagen fibrils were three-dimensionally reconstructed using the computer graphic imaging process. Findings from this study suggest that there exist a local "bulging" along the fibrils corresponding to the 680 A periodicity in which additional mass of minerals were observed to be accommodated within the collagen fibril structure at this sites.     

The importance of size-exclusion characteristics of type I collagen in bonding to dentin matrices

The mineral phase of dentin is located primarily within collagen fibrils. During development, bone or dentin collagen fibrils are formed first and then water within the fibril is replaced with apatite crystallites. Mineralized collagen contains very little water. During dentin bonding, acid-etching of mineralized dentin solubilizes the mineral crystallites and replaces them with water. During the infiltration phase of dentin bonding, adhesive comonomers are supposed to replace all of the collagen water with adhesive monomers that are then polymerized into copolymers. The authors of a recently published review suggested that dental monomers were too large to enter and displace water from collagen fibrils. If that were true, the endogenous proteases bound to dentin collagen could be responsible for unimpeded collagen degradation that is responsible for the poor durability of resin–dentin bonds. The current work studied the size–exclusion characteristics of dentin collagen, using a gel-filtration-like column chromatography technique, using dentin powder instead of Sephadex. The elution volumes of test molecules, including adhesive monomers, revealed that adhesive monomers smaller than ～1000 Da can freely diffuse into collagen water, while molecules of 10,000 Da begin to be excluded, and bovine serum albumin (66,000 Da) was fully excluded. These results validate the concept that dental monomers can permeate between collagen molecules during infiltration by etch-and-rinse adhesives in water-saturated matrices.     

New developments in polymer-controlled,bioinspired calcium phosphate mineralization from aqueous solution

The polymer-controlled and bioinspired precipitation of inorganic minerals from aqueous solution at near-ambient or physiological conditions avoiding high temperatures or organic solvents is a key research area in materials science. Polymer-controlled mineralization has been studied as a model for biomineralization and for the synthesis of (bioinspired and biocompatible) hybrid materials for a virtually unlimited number of applications. Calcium phosphate mineralization is of particular interest for bone and dental repair. Numerous studies have therefore addressed the mineralization of calcium phosphate using a wide variety of low- and high-molecular-weight additives. In spite of the growing interest and increasing number of experimental and theoretical data, the mechanisms of polymer-controlled calcium phosphate mineralization are not entirely clear to date, although the field has made significant progress in the last years. A set of elegant experiments and calculations has shed light on some details of mineral formation, but it is currently not possible to preprogram a mineralization reaction to yield a desired product for a specific application. The current article therefore summarizes and discusses the influence of (macro)molecular entities such as polymers, peptides, proteins and gels on biomimetic calcium phosphate mineralization from aqueous solution. It focuses on strategies to tune the kinetics, morphologies, final dimensions and crystal phases of calcium phosphate, as well as on mechanistic considerations.     

可降解生物材料聚乳酸-羟基乙酸仿生矿化的实验研究

目的：通过对聚乳酸-羟基乙酸共聚物(poly lactide-co-glycolide，PLGA)的仿生矿化，表面改性，以提高其细胞粘附性；探讨影响仿生矿化的因素和条件，为进一步制备组织工程化人工骨提供依据和实验基础。方法：PLGA膜经碱性溶液水解处理后，应用高温显微镜测量材料表面润湿角的变化；碱处理后的PLGA膜和三维多孔PLGA分别在模拟体液(Simulated Body Fluid，SBF)中矿化14d，在1．5倍SBF中矿化9d，应用扫描电镜进行矿化物形貌观察，X射线能谱分析钙磷比值，X射线衍射仪和傅立叶转换红外光谱仪行矿化物物相分析。结果：PLGA经碱性溶液水解处理后表面亲水性明显增强，在SBF及1．5倍SBF中矿化后表面可以形成明显的矿化物；矿化物的形态与矿化液的浓度有关；矿化物主要成分为羟基磷灰石(HA)，含有碳酸根成分，钙磷比为1．53，类似于人骨无机质。结论：PLGA仿生矿化是制备结构及性质类似骨基质人工骨的可行方法。     

Lactic acid based PEU/HA and PEU/BCP composites: Dynamic mechanical characterization of hydrolysis

Surface structure in the form of roughness and organized patterning can affect osteoblastic adhesion and proliferation. This study investigates the effect of reconstituted collagen fibrils on the deposition pattern of a homogeneous inorganic mineral (sodium chloride). The patterns were monitored from nanometer to millimeter scales using atomic force and light microscopies. Initially, mineral deposits formed blocks following the contour of the collagen fibrils. At later times, dendritic structures formed. This demonstrates that collagen fibrils can affect the surface deposition pattern of saline minerals. It is also shown that collagen fibril diameter and the stoichiometry of the inorganic and organic phases effect the surface distribution of minerals.