Carbonated Calcium Hydroxyapatite Research Articles

Unraveling the structural complexity of and the effect of calcination temperature on calcium phosphates derived from Oreochromis niloticus bones

In this study, the interplay between the structural complexity, microstructure, and mechanical properties of calcium phosphates (CaPs) derived from fish bones, prepared at various calcination temperatures, and their corresponding sintered ceramics was explored. Fourier-transform infrared analysis revealed that the calcined powders primarily consisted of hydroxyapatite (HAp) and carbonated calcium hydroxyapatite, with an increasing concentration of Mg-substituted β-tricalcium phosphate (β-TCP) as the calcination temperature was increased. X-ray diffraction patterns showed enhanced sharpness of the peaks at higher temperatures, indicating a larger crystallite size and improved crystallinity. The ceramics exhibited a significantly larger crystallite size and an increased concentration of the β-TCP phase. Rietveld analysis revealed a larger volume of the β-TCP phase in the ceramics than in their calcined powders; this could be attributed to a newly formed β-TCP phase due to the decomposition of HAp. Extended X-ray absorption fine structure analysis revealed the incorporation of Mg in the Ca2 site of HAp, Ca2 site of β-TCP, and Ca5 site of β-TCP, with a higher substitution of Mg in the Ca5 site of β-TCP at elevated temperatures. The mechanical properties of HAp ceramics can be improved by increasing the calcination temperature because of their improved relative density and dense porous structure at elevated temperatures. This comprehensive investigation sheds light on the phase evolution, microstructural changes, and consequential impact on the mechanical properties of CaPs derived from fish bones, thereby facilitating the development of tailored CaP ceramics for biomedical applications.

MASS PRODUCTION OF OSTEOGENICALLY ENHANCED HA/CC SCAFFOLDS FOR BONE REPAIR

Accidents, osteoporosis or cancer can cause severe bone damage requiring grafts to heal. All current grafting methods have disadvantages including scarcity and infection/rejection risks. An alternative is therefore needed. Hydroxyapatite/calcium carbonate (HA/CC) scaffolds mimic the mineral bone composition but lack growth factors present in auto- and allografts, limiting their osteoinductive capacity. We hypothesize that this will increase the osteogenicity and osteoinductivity of scaffolds through the presence of growth factors. The objectives of this study are to develop and mass-produce grafts with enhanced osteoinductive capacity.HA/CC scaffolds were cultured together with umbilical cord mesenchymal stem cells in bioreactors so that they adhere to the surface and deposit growth factors. Cells growing on the scaffolds are confirmed by Alamar blue assays, SEM, and confocal microscopy. ELISA and IHC are used to assess the growth factor content of the finished product.It has been confirmed that cells attach to the scaffolds and proliferate over time when grown in bioreactors. Dynamic seeding of cells is clearly advantageous for cell deposits, equalizing the amount of cells on each scaffold granule.Hydroxyapatite/calcium carbonate scaffolds support cell-growth. This should be confirmed by further research, including Quantification of BMPs and other indicators of osteogenic differentiation such as Runx2, osteocalcin and ALP is pending, and amounts are expected to be increased in enhanced scaffolds and in-vivo implantation.

Synthesis and Characterization of Nano-sized Carbonated Calcium Hydroxyapatite (CHAp) from Rebon shrimp (Acetes erythraeus) as a Candidate for Dental Restoring Application

Carbonated calcium hydroxyapatite (CHAp) exhibits excellent biocompatibility with bone and teeth, making it an ideal candidate for orthopedic and dental application. However, the study of CHAp synthesis from natural material is still scarce. The purpose of this research is to synthesize and characterize of CHAp, using Rebon shrimp (Acetes erythraeus) as a calcium source. The synthesis was conducted by hydrothermal method with the variation of Ca/P ratios 1.61; 1.67; 1.73. The as-prepared CHAp was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and scanning electron microscopy-energy dispersive X-ray (SEM-EDX). The FT-IR results show that synthesized material exhibited characteristic CHAp band of hydroxide at 3448 and 1635 cm-1, carbonate at 872 and 1427 cm-1, and phosphate at 1049; 606; and 570 cm-1. The diffractogram pattern assigned the all observed peak of CHAp are in good agreement compared to CHAp database with the nano-scale size. It also observed that the high Ca/P ratio will decrease the crystallinity of CHAp. The as-prepared CHAp micrograph is agglomerates spherical form with size between 5-20 nm which build up from 18–26 nm crystallite particles. The result of this research confirmed that Rebon shrimp is the promising materials for calcium source in CHAp production.

Nanocrystalline Calcium Carbonate Hydroxyapatites Containing Multiwall Carbon Nanotubes: Synthesis and Physicochemical Characterization

Nanocomposites (NCs) based on carbonated calcium hydroxyapatite (CHA) (bioapatite, an analogue of the inorganic component of mammalian bone tissue), carbonate apatite (Ca10(PO4)6CO3, CA), and multiwall carbon nanotubes (CNTs) are prepared in the system CaCl2–(NH4)2HPO4–NH4HCO3–NH3–CNT–H2O (25°C) by coprecipitation of calcium and phosphorus salts with CNTs from aqueous solutions. The physicochemical properties of nanocomposites are studied as dependent on their formation conditions and composition using the solubility (residual concentrations) method and pH measurements. The composition, crystal structure, morphology, spectroscopic and thermal characteristics of the synthesized CHA/CNT and CA/CNT NCs are determined using chemical analysis, X-ray powder diffraction, thermal analysis, and IR spectroscopy. Either CHA/CNT NCs of composition Ca10(PO4)6(CO3)x(OH)2–2х · yCNT · zH2O, where х = 0.2; 0.5; 0.8; y = 1, 2, 3; z = 6.8–10.8, or (when х = 1) CA/CNT NCs of composition Ca10(PO4)6CO3 · yCNT · zH2O, where y = 1–3; z = 6.9–10.8, are formed as the carbonate and CNT contents of the NC increase. Our results favor the understanding of the effect of carbonization and CNTs on the metabolic formation of native bone tissue apatite and can be used for the design of efficient ceramics for bone implants.

The hydroxyapatite Turkish Delight: a technical note

Nasal dorsum augmentation is commonly performed using autologous cartilage grafts, also in the Turkish Delight technique. The aim of this study was to describe a modification of the Turkish Delight technique for dorsal augmentation consisting of small hydroxyapatite-calcium carbonate granules (0.5–1 mm) that were wrapped in layers of oxidized cellulose and glued with 1–2-cm3 fibrin sealant and to compare its utility with that of other techniques. Clinically stable and satisfactory results were achieved in the four cases examined. Cone-beam computerized tomography (CBCT) imaging revealed that there was no degradation of the graft up to 2 years after surgery. The use of a modified Turkish Delight method using hydroxyapatite granules promises to be a valuable option for the correction of nasal dorsum deficiency.

Chemical and Structural Information from the Enamel of a Troodon Tooth Leading to an Understanding of Diet and Environment.

Synchrotron micro X-ray fluorescence (XRF) spectroscopy with two-dimensional element mapping, micro X-ray diffraction (XRD), electron spin resonance spectroscopy (ESR) and atomic force microscopy (AFM) were used to investigate the chemical and structural nature of the enamel of a tooth from Troodon, a small theropod dinosaur. These methods show that the crystallites in the Troodon tooth are submicron-sized carbonated calcium hydroxyapatite, which are semi-randomly oriented with a preferred orientation of (002) towards the surface of the tooth. Transition metal ions are distributed in the voids between crystallite clusters. Comparison of the ESR spectra indicates that the Troodon tooth had less exposure to UV than a fossilized crocodile tooth.

An Approach to Breast Cancer Diagnosis via PET Imaging of Microcalcifications Using (18)F-NaF.

Current radiologic methods for diagnosing breast cancer detect specific morphologic features of solid tumors or any associated calcium deposits. These deposits originate from an early molecular microcalcification process of 2 types: type 1 is calcium oxylate and type II is carbonated calcium hydroxyapatite. Type I microcalcifications are associated mainly with benign tumors, whereas type II microcalcifications are produced internally by malignant cells. No current noninvasive in vivo techniques are available for detecting intratumoral microcalcifications. Such a technique would have a significant impact on breast cancer diagnosis and prognosis in preclinical and clinical settings. (18)F-NaF PET has been used solely for bone imaging by targeting the bone hydroxyapatite. In this work, we provide preliminary evidence that (18)F-NaF PET imaging can be used to detect breast cancer by targeting the hydroxyapatite lattice within the tumor microenvironment with high specificity and soft-tissue contrast-to-background ratio while delineating tumors from inflammation. Mice were injected with approximately 10(6) MDA-MB-231 cells subcutaneously and imaged with (18)F-NaF PET/CT in a 120-min dynamic sequence when the tumors reached a size of 200-400 mm(3). Regions of interest were drawn around the tumor, muscle, and bone. The concentrations of radiotracer within those regions of interest were compared with one another. For comparison to inflammation, rats with inflamed paws were subjected to (18)F-NaF PET imaging. Tumor uptake of (18)F(-) was significantly higher (P < 0.05) than muscle uptake, with the tumor-to-muscle ratio being about 3.5. The presence of type II microcalcification in the MDA-MB-231 cell line was confirmed histologically using alizarin red S and von Kossa staining as well as Raman microspectroscopy. No uptake of (18)F(-) was observed in the inflamed tissue of the rats. Lack of hydroxyapatite in the inflamed tissue was verified histologically. This study provides preliminary evidence suggesting that specific targeting with (18)F(-) of hydroxyapatite within the tumor microenvironment may be able to distinguish between inflammation and cancer.

Effect of processing conditions on the crystallinity and structure of carbonated calcium hydroxyapatite (CHAp)

Previous synthesis routes created apatites in low crystallinity and high crystallinity states, but a wider range will extend the design capabilities of apatites for hard tissue replacements. While high crystallinity apatites are more conventional, this work investigated lower crystallinity variations from an amorphous state to low crystallinity apatite. Carbonated hydroxyapatite was prepared by precipitating an amorphous phase followed by crystallization at 650 °C at slow (5 °C min−1) and fast heating rates (60 °C min−1). The effect of processing conditions on crystallinity and structural changes was evaluated by thermal analysis, X-ray diffraction, transmission electron microscopy, Fourier transform infrared and Raman spectroscopy. Furthermore, peak deconvolution of IR and Raman spectra resolved the carbonate and phosphate bands and revealed the carbonate and crystalline phase content in CHAp. Similar to precipitation of crystalline apatites, the crystallization at elevated temperature led to carbonate in both the phosphate and hydroxyl positions. Heating at 650 °C provided a nanosized spherical hydroxyapatite containing carbonate controlled by the heating rate. This creates a mechanism for creating a large range in crystallinity with a greater resorption capability for regenerative medicine.

Fractionated-combustion analysis of carbonate-containing phases in composite materials of the hydroxyapatite-calcium carbonate system

Materials in the hydroxyapatite (HA)-calcium carbonate (CC) system were synthesized by a precipitation method from aqueous solutions. According to the data of X-ray phase analysis and IR spectroscopy, the powders consisted of CC and AB-type carbonate-substituted HA (CHA). In order to determine the content of carbonate-containing phases in materials, the temperature-temporal mode of fractionated-combustion analysis of carbon was developed. The quantitative phase ratios and the degree of substitution of carbonate groups in CHA were determined. It was shown that the degree of substitution of carbonate groups in CHA increased from 2.47 to 5.31 wt % as the CC content increased from 13.50 to 88.33 wt %.

Preparation and structure of carbonated calcium hydroxyapatite substituted with heavy rare earth ions

Calcium hydroxyapatite (CaHap) particles substituted five types of heavy rare earth ions (Ln: Y3+, Gd3+, Dy3+, Er3+ and Yb3+) were synthesized using a precipitation method and characterized using various means. These Ln ions strongly affected the crystal phases and the structures of the products. With increasing Ln/(Ln+Ca) in the starting solution ([XLn]), the length and the crystallinity of the particles first increased and then decreased. The rare earth metal-calcium hydroxyapatite (LnCaHap) solid solution particles were obtained at [XY]≤0.10 for substituting Y system and at [XLn]≤0.01–0.03 for substituting the other Ln systems. LnPO4 was mixed with LnCaHap at higher [XLn] for all Ln systems. A series of yttrium-calcium hydroxyapatite (YCaHap) solid solutions with [XY]=0–0.10 were investigated using XRD, TEM, ICP-AES, IR and TG–DTA in detail.

The effect of composition of calcium phosphate composite scaffolds on the formation of tooth tissue from human dental pulp stem cells

Different approaches towards making 3-dimensional (3-D) bioengineered tooth for future replacement therapy have been developed including scaffold-based tooth regeneration. However, selection of optimal scaffold for future clinical application remains a challenge. In the present study, we tested biocompatibility of four different types of 3-D scaffolds for tooth-tissue regeneration, including a pure poly(lactide-co-glycolide) (PLGA) (70/30, mol/mol) scaffold and three types of calcium phosphate contained composites scaffolds that were 50 wt% of PLGA combined with 50 wt% of hydroxyapatite (HA), tricalcium phosphate (TCP) or calcium carbonate hydroxyapatite (CDHA) respectively. These scaffolds were fabricated by the particle leaching in combination with phase separation technology. Surface modification of these scaffolds was further performed by an ammonia plasma treatment and anchorage of collagen technology. Effect of composition of the composite scaffolds on proliferation of human dental pulp stem cells (DPSCs) was accessed using in vitro MTT assay and in vivo BrdU labeling. Differentiation capability of the DPSCs in the scaffolds was analyzed by measurement of the levels of calcified tissue formation and ALP activity. Our results showed that while the calcium phosphate contained compound is able to support regeneration of tooth tissue effectively, the PLGA/TCP scaffold is more appropriate for the proliferation and differentiation of DPSCs. Furthermore, seeding of dissociated 4-dpn rat tooth bud cells on the PLGA/TCP scaffold generated dentin- and pulp-like tissues. Our results demonstrate that the PLGA/TCP scaffold is superior to the other three scaffolds for tooth-tissue regeneration, especially for dentin formation.

The induction of bone formation by coral-derived calcium carbonate/hydroxyapatite constructs

The spontaneous induction of bone formation in heterotopic rectus abdominis and orthotopic calvarial sites by coral-derived biomimetic matrices of different chemical compositions was investigated in a long-term study in the non-human primate Papio ursinus. Coral-derived calcium carbonate constructs were converted to hydroxyapatite by hydrothermal exchange. Limited conversion produced hydroxyapatite/calcium carbonate (HA/CC) constructs of 5% and 13% hydroxyapatite. Rods of 20 mm in length and 7 mm in diameter were implanted in heterotopic rectus abdominis sites; discs 25 mm in diameter were implanted in orthotopic calvarial defects of six adult non-human primates P. ursinus. Heterotopic samples also included fully converted hydroxyapatite replicas sintered at 1100 °C. To further enhance spontaneous osteoinductive activity, fully converted hydroxyapatite replicas were coated with the synthetic peptide P15 known to increase the adhesion of fibroblasts to anorganic bovine mineral. Bone induction was assessed at 60, 90 and 365 days by histological examination, alkaline phosphatase and osteocalcin expression, as well as by the expression of BMP-7, GDF-10 and collagen type IV mRNAs. Induction of bone occurred in the concavities of the matrices at all time points. At 365 days, bone marrow was evident in the P15-coated and uncoated implants. Resorption of partially converted calcium carbonate/hydroxyapatite was apparent, as well as remodeling of the newly formed bone. Northern blot analyses of samples from heterotopic specimens showed high levels of expression of BMP-7 and collagen type IV mRNA in all specimen types at 60 days, correlating with the induction of the osteoblastic phenotype in invading fibrovascular cells. Orthotopic specimens showed prominent bone formation across the different implanted constructs. The concavities of the matrices biomimetize the remodeling cycle of the osteonic primate cortico-cancellous bone and promote the ripple-like cascade of the induction of bone formation. This study demonstrates for the first time that partially converted HA/CC constructs also induce spontaneous differentiation of bone, albeit only seen one year post-implantation.

Hydrothermal synthesis and nanostructure of carbonated calcium hydroxyapatite

The influence of precursor concentration, pressure, temperature and time of hydrothermal synthesis on the development of calcium hydroxyapatite structure has been analyzed. The obtained results show that it is possible to adjust the conditions of hydrothermal synthesis from solutions of relatively high concentrations to obtain calcium hydroxyapatite nanopowders of well-defined structure. The relationship between the synthesis and the lattice parameters, as well as the crystallite size and the microstructure of synthesized hydroxyapatite has been established. The synthesized powders are preferentially carbonated hydroxyapatite of the B type in the form of agglomerates that accommodate two-modal size pores of 1.5-10 and 50-200 nm. The structure of calcium hydroxyapatite particles consists of crystallites 8-22 nm in size, bound within prime particles, which size is between 10 and 63 nm, that in turn form bigger agglomerates 200 nm in size, which further cluster building up agglomerates 5-20 microm in size.

Biocompatibility of nanostructured carbonated calcium hydroxyapatite obtained by hydrothermal method

Evaluation of biomaterials as safe and effective therapies needs preclinical models to estimate their biologic potential. This paper investigates biocompatibility by the in vivo assessment of the muscle tissue reaction after implantation of the hidrothermally produced calciumhydroxyapatite. Specific attention has to be given to the synthesis technique which influences the stereology of the material and the behavior of the material in living tissues. The synthesized powders of hydroxyapatite are preferentially carbonated hydroxyapatite of the B type in the form of agglomerates that accommodate two-modal size pores of 1.5-10 nm and 50-200 nm. The particles are built from crystallites of 8-22 nm in size, bind inside of the prime particles sized is between 10 and 63 nm. They form agglomerates of 200 nm in size and these were further clustered building up the biggest agglomerates of 5- 20 _m. Biocompatibility assessment revealed that only mild to moderate inflammatory reaction was seen around the calciumhydroxyapatite implants. Calciumhydroxyapatite failed to show any substantional toxicity.

Preparation and characterization of carbonated barium–calcium hydroxyapatite solid solutions

Particles of carbonated barium–calcium hydroxyapatite solid solutions (BaCaHap) with different Ba/(Ba + Ca) ( X Ba ) atomic ratios were prepared by a wet method at 100 °C and characterized by various means. The crystal phases and structures of the products strongly depended on the composition of the starting solution, that is, the Ba/(Ba + Ca) atomic ratio ( [ X Ba ] ) and H 3PO 4 concentration ([H 3PO 4]) in the solution. BaCaHap with X Ba ⩽ 0.43 could be prepared at [ X Ba ] ⩽ 0.7 by changing [H 3PO 4], but could never be obtained at [ X Ba ] = 0.8 – 0.95 regardless of [H 3PO 4]. The carbonated calcium hydroxyapatite particles prepared at [ X Ba ] = 0 were fine and short rod-shaped particles (ca. 14 × 84 nm). With increasing [ X Ba ] from 0 to 0.8, the particles obtained became large spherical agglomerates. The carbonated barium hydroxyapatite particles formed at [ X Ba ] = 1 were long rod-shaped agglomerates (ca. 0.2 × 2 μm) of fine primary particles. The amount of CO 2 adsorbed irreversibly on a series of BaCaHaps showed a minimum at (Ba + Ca)/(P + C) atomic ratio of around 1.56, which agreed well with the minimum cation/P ratio obtained for the other hydroxyapatites, as already reported.

A resorbable porous ceramic composite bone graft substitute in a rabbit metaphyseal defect model

The success of converted corals as a bone graft substitute relies on a complex sequence of events of vascular ingrowth, differentiation of osteoprogenitor cells, bone remodeling and graft resorption occurring together with host bone ingrowth into and onto the porous coralline microstructure or voids left behind during resorption. This study examined the resorption rates and bone infiltration into a family of resorbable porous ceramic placed bilaterally in critical sized defects in the tibial metaphyseal–diaphyseal of rabbits. The ceramics are made resorbable by partially converting the calcium carbonate of corals to form a hydroxyapatite (HA) layer on all surfaces. Attempts have been made to control the resorption rate of the implant by varying the HA thickness. New bone was observed at the periosteal and endosteal cortices, which flowed into the centre of the defect supporting the osteoconductive nature of partially converted corals. The combination of an HA layer and calcium carbonate core provides a composite bone graft substitute for new tissue integration. The HA–calcium carbonate composite demonstrated an initial resorption of the inner calcium carbonate phase but the overall implant resorption and bone ingrowth behaviour did not differ with HA thickness.

New bioactive, degradable composite microspheres as tissue engineering substrates.

Novel bioactive, degradable polymer/glass/ceramic composite microspheres were developed using a solid-in-oil-in-water (s/o/w) emulsion solvent removal method. Modified bioactive glass (MBG) powders were encapsulated into the polylactic acid (PLA) matrix. Scanning electron microscopy and energy-dispersive X-ray analyses revealed that the MBG powders were mostly embedded in the polymer matrix, and submicron-size pores were present at the surface. Immersion in simulated physiological fluid (SPF) was used to evaluate the surface reactivity of the microspheres. The polymeric surface was fully transformed into carbonated calcium hydroxyapatite (c-HA) after 3 weeks of immersion. In contrast, PLA microspheres showed no evidence of any calcium phosphate deposition. Ion concentration analyses revealed a decrease in Ca and P concentrations and an increase in Si concentration in the SPF immersed with composite microspheres during the 3-week period. The Ca and P uptake rates decreased after 2 days of incubation. This coincided with the decrease of the Si release rate. These data lend support to the suggestion that the Si released from the MBG powders present in the polymer matrix is involved in the formation of the Ca-P layer. Our results support the concept that these new bioactive, degradable composite microspheres may serve as microcarriers for synthesis of bone and other tissues in vitro and in vivo.

Chemical preparation of carbonated calcium hydroxyapatite powders at 37°C in urea-containing synthetic body fluids

An important inorganic phase of synthetic bone applications, calcium hydroxyapatite (HA, Ca 10(PO 4) 6(OH) 2), was prepared as a single-phase ceramic powder. Carbonated HA powders were formed from calcium nitrate tetrahydrate and di-ammonium hydrogen phosphate salts dissolved in aqueous ‘synthetic body fluid’ (SBF) solutions, containing urea (H 2NCONH 2), at 37 °C and pH of 7·4, by using a novel chemical precipitation technique. These powders were also found to contain trace amounts of Na and Mg impurities in them, originated from the use of SBF solutions, instead of pure water, during their synthesis. The characterization and chemical analysis of the synthesized powders were performed by powder X-ray diffraction (XRD), Fourier-transformed infra-red spectroscopy (FT–IR), and inductively-coupled plasma atomic emission spectroscopy (ICP–AES).

Bonelike Coatings onto Ceramics by Reactive Magnetron Sputtering

Bonelike coating of partially carbonated, calcium hydroxyapatite onto strong ceramics and metals is one of the current objectives in biomaterials science for the development of bioactive implant materials. We recently accomplished bonelike apatite coating onto ceramic alumina by the newly developed reactive rf‐magnetron sputtering method, which was undertaken in a mixed CO2/Ar gas. The key of this method is the use of calcium phosphate glass targets with much lower Ca/P ratios than the stoichiometric value of hydroxyapatite. Surface analyses by XRD, EDX, and FT‐IR identified the formation of bonelike carbonated calcium hydroxyapatite, in which CO2‐3 ions occupied the lattice positions of PO3‐4. The bioactivity of the coated ceramic materials was confimed by in vitro experiments on the bonelike crystal growth on their surfaces in a simulated body fluid, and the growth rate on the coated layers was experimentally proved to be much faster than that on uncarbonated hydroxyapatite

Molecular mechanisms of dental enamel formation.

Tooth enamel is a unique mineralized tissue in that it is acellular, is more highly mineralized, and is comprised of individual crystallites that are larger and more oriented than other mineralized tissues. Dental enamel forms by matrix-mediated biomineralization. Enamel crystallites precipitate from a supersaturated solution within a well-delineated biological compartment. Mature enamel crystallites are comprised of non-stoichiometric carbonated calcium hydroxyapatite. The earliest crystallites appear suddenly at the dentino-enamel junction (DEJ) as rapidly growing thin ribbons. The shape and growth patterns of these crystallites can be interpreted as evidence for a precursor phase of octacalcium phosphate (OCP). An OCP crystal displays on its (100) face a surface that may act as a template for hydroxyapatite (OHAp) precipitation. Octacalcium phosphate is less stable than hydroxyapatite and can hydrolyze to OHAp. During this process, one unit cell of octacalcium phosphate is converted into two unit cells of hydroxyapatite. During the precipitation of the mineral phase, the degree of saturation of the enamel fluid is regulated. Proteins in the enamel matrix may buffer calcium and hydrogen ion concentrations as a strategy to preclude the precipitation of competing calcium phosphate solid phases. Tuftelin is an acidic enamel protein that concentrates at the DEJ and may participate in the nucleation of enamel crystals. Other enamel proteins may regulate crystal habit by binding to specific faces of the mineral and inhibiting growth. Structural analyses of recombinant amelogenin are consistent with a functional role in establishing and maintaining the spacing between enamel crystallites.