[Show abstract][Hide abstract] ABSTRACT: Vertebrate mineralized tissues, i.e., enamel, dentin, cementum, and bone, have unique hierarchical structures and chemical compositions. Although these tissues are similarly comprised of a crystalline calcium apatite mineral phase and a protein component, they differ with respect to crystal size and shape, level and distribution of trace mineral ions, the nature of the proteins present, and their relative proportions of mineral and protein components. Despite apparent differences, mineralized tissues are similarly derived by highly concerted extracellular processes involving matrix proteins, proteases, and mineral ion fluxes that collectively regulate the nucleation, growth and organization of forming mineral crystals. Nature, however, provides multiple ways to control the onset, rate, location, and organization of mineral deposits in developing mineralized tissues. Although our knowledge is quite limited in some of these areas, recent evidence suggests that hard tissue formation is, in part, controlled through the regulation of specific molecules that inhibit the mineralization process. This paper addresses the role of mineralization inhibitors in the regulation of biological mineralization with emphasis on the relevance of current findings to the process of amelogenesis. Mineralization inhibitors can also serve to maintain driving forces for calcium phosphate precipitation and prevent unwanted mineralization. Recent evidence shows that native phosphorylated amelogenins have the capacity to prevent mineralization through the stabilization of an amorphous calcium phosphate precursor phase, as observed in vitro and in developing teeth. Based on present findings, the authors propose that the transformation of initially formed amorphous mineral deposits to enamel crystals is an active process associated with the enzymatic processing of amelogenins. Such processing may serve to control both initial enamel crystal formation and subsequent maturation.
Frontiers in Physiology 09/2014; 5:339. DOI:10.3389/fphys.2014.00339 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Our previous in vitro studies have shown that recombinant full-length porcine amelogenin rP172 can transiently stabilize amorphous calcium phosphate (ACP) and uniquely guide the formation of well-aligned bundles of hydroxyapatite (HA) crystals, as seen in the secretory stage of amelogenesis. This functional capacity is dependent on the hydrophilic C-terminal domain of full-length amelogenin. However, we have also found that native phosphorylated (single S-16 site) forms of full-length (P173) and C-terminal cleaved (P148) amelogenins can stabilize ACP for > 2 d and prevent HA formation. The present study was carried out to test the hypothesis that, at reduced concentrations, native full-length P173 also has the capacity to guide ordered HA formation. The effect of P148 and P173 concentrations (0.2-2.0 mg/ml) on the rate of spontaneous calcium phosphate precipitation was monitored via changes in solution pH, while mineral phases formed were assessed using TEM. At higher P173 concentrations (1.0-2.0 mg/ml), limited mineral formation occurred and only ACP nanoparticles were observed during a 48 h period. However, at 0.4 mg/ml P173, a predominance of organized bundles of linear, needle-like HA crystals were observed. At 0.2 mg/ml of P173, limited quantities of less organized HA crystals were found. Although P148 similarly stabilized ACP, it did not guide ordered HA formation, like P173. Hence, the establishment of the hierarchical enamel structure during secretory stage amelogenesis may be regulated by the partial removal of full-length amelogenin via MMP20 proteolysis, while predominant amelogenin degradation products, like P148, serve to prevent uncontrolled mineral formation.
Connective Tissue Research 08/2014; 55(S1):21-24. DOI:10.3109/03008207.2014.923853 · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine amelogenins undergo step-wise self-assembly. Although the overall structural organization of P173 and rP172 oligomers was similar and resembled oligomers of murine recombinant amelogenin rM179, there were subtle differences suggesting that a single phosphorylated serine present in P173 might affect amelogenin self-assembly. Our mineralization studies demonstrated that both P173 and rP172 oligomers stabilize initial mineral clusters. Importantly, however, rP172 regulated the organization of initial mineral clusters into linear chains and guided the formation of parallel arrays of elongated mineral particles, which are the hallmark of enamel structural organization. These results are similar to those obtained previously using full-length recombinant murine amelogenin (Fang et al., 2011a). In contrast to that seen with rP172, phosphorylated P173 strongly inhibits mineralization for extended periods of time. We propose that these differences might be due to the differences in the structural organization and charge distribution between P173 and rP172. Overall our studies indicate that self-assembly of amelogenin and the mechanisms of its control over mineralization might be universal across different mammalian species. Our data also provide new insight into the effect of phosphorylation on amelogenin self-assembly and its regulation of mineralization.
[Show abstract][Hide abstract] ABSTRACT: Recent studies in our laboratory have shown that phosphorylated full-length (P173, 25 kDa) and cleaved (P148, 20-kDa) porcine amelogenins inhibit spontaneous calcium phosphate crystal formation in vitro, by stabilizing an amorphous calcium phosphate precursor phase (ACP). Since amelogenin undergoes proteolysis by MMP20 soon after secretion, its effect and the effect of specific amino acid domains within amelogenin on calcium phosphate formation is of great interest. Objectives: To test the hypothesis that ACP transformation into bundles of ordered enamel-like hydroxyapatite (HA) crystals can be induced by MMP20 proteolysis of full-length P173. Methods: Calcium and phosphate were sequentially added to protein solutions (2 mg/ml, 60 mL, ~pH 4) with MMP-20 (at ratios of 100:1 and 200:1) to yield 2.5 mM calcium and 1.5 mM phosphate at physiological ionic strength (163 mM) in 50 mM Tris-HCl, pH 7.4 at 37oC. Protein degradation with time was assessed by gel-electrophoresis and mineral products formed were characterized by TEM and SAED. Results: MMP20 was found to cleave P173 to primarily generate 23, 20, 13, and 11-kDa fragments, corresponding to P162, P148, P62 and P63, while limited P148 digestion was observed. With P173 and added MMP20, a slight pH drop (DpH ~1.0) was observed after 12 and 24 hr (P173:MMP20 = 100:1 and 200:1, respectively), along with the formation of bundles of needle-like HA crystals. Such pH decreases were not seen in controls (i.e., without MMP20) or in the presence of P148 with added MMP20, corresponding to the sole formation ACP nanoparticles. Conclusions: Results obtained demonstrate that P173 cleavage by MMP20 can induce the transformation of stabilized ACP into ordered bundles of HA crystals. These findings suggest that the formation of the hierarchical enamel structure may be regulated by the proteolysis of full-length native amelogenin, during the early stages of enamel formation. Supported by NIDCR grant R01-DE016376.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: New insights into amelogenesis will be provided through the presentation of key findings on the effect of amelogenin phosphorylation on protein self-assembly and the regulation of spontaneous calcium phosphate formation in vitro. Evidence will show that native amelogenins with a single phosphorylated site are potent stabilizers of amorphous calcium phosphate nanoparticles and inhibit their transformation to apatitic crystals. Amelogenin phosphorylation was also found to induce functionally important conformational changes that affect both protein-mineral and protein - mineral ion interactions. Importantly, the observed inhibition of crystal formation by full-length native amelogenin can be removed through its proteolysis resulting in the formation of bundles of enamel-like crystals. These findings are consistent with reported data showing that the same amorphous to crystalline mineral transformation occurs in vivo during initial stages of enamel formation. Results suggest that amelogenin phosphorylation and regulation of mineral phase transformation are essential elements of the enamel formation process.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: Dental enamel is made of arrays of elongated apatitic crystallites organized into an intricate three-dimensional microstructure. Amelogenin, the major enamel protein, regulates growth and organization of the enamel mineral. Our recent cryo-TEM study of recombinant mouse amelogenin (rM179) revealed that it undergoes step-wise self-assembly. Furthermore, rM179 oligomers stabilize mineral pre-nucleation clusters and guide their arrangement, prior to crystallization. Interestingly, native porcine amelogenin containing a single phosphoserine, inhibits calcium phosphate crystallization. To test the hypothesis that step-wise assembly is a universal trait of amelogenins, and to study the effects of phosphorylation on amelogenin self-assembly and its effect on biomineralization, we have conducted cryo-TEM studies of porcine native phosphorylated (P173) and recombinant nonphosphorylated (rP172) amelogeinins.
Method: P173 and rP172 were assembled in aqueous solutions at pH=8.0 at 37ºC. Droplets of the solutions were mounted on lacey carbon grids and incubated from 1 to 30 min. To induce mineralization, CaCl2 and Na2HPO4 were added to the reaction and the samples were incubated at 37ºC, pH~7.6 from 10 mins to 2 hrs. The grids were vitrified in liquid ethane and studied in cryo-TEM under low-dose conditions. Single-particle reconstructions were performed to explore the assembly process.
Result: Both rP172 and P173 undergo step-wise assembly, with the monomers arranging into cage-like oligomers which in turn form nanospheres. Our mineralization studies demonstrated that P173 and rP172 oligomers stabilize mineral pre-nucleation clusters for more than 2 hrs and P173 has the stronger inhibition capacity than rP172. The pre-nucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites.
Conclusion: Our cryo-TEM studies have revealed that porcine amelogenins undergo step-wise self-assembly similarly to murine amelogenin and that the inhibition of calcium phosphate mineralization occurs via stabilization of pre-nucleation clusters.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Lhx6 is a LIM-homeobox transcription factor expressed during embryogenesis, however the molecular mechanisms regulating Lhx6 transcriptional activities are unknown. Lhx6 and the Pitx2 homeodomain transcription factor have overlapping expression patterns during tooth and craniofacial development and in this report we demonstrate new transcriptional mechanisms for these factors. Pitx2 and Lhx6 are co-expressed in the oral and dental epithelium and epithelial cell lines. Lhx6 expression is increased in Pitx2c transgenic mice and decreased in Pitx2 null mice. PITX2 activates endogenous Lhx6 expression and the Lhx6 promoter while Lhx6 represses its promoter activity. Chromatin immunoprecipiation experiments reveal endogenous Pitx2 binding to the Lhx6 promoter. Lhx6 directly interacts with PITX2 to inhibit PITX2 transcriptional activities and activation of multiple promoters. Bimolecular fluorescence complementation assays reveal an Lhx6-Pitx2 nuclear interaction in living cells. Lhx6 has a dominant repressive effect on the PITX2 synergistic activation with Lef-1 and β-catenin co-factors. Thus, Lhx6 acts as a transcriptional repressor and represses the expression of several genes involved in odontogenesis. We have identified specific defects in incisor, molar, mandible, bone and root development and late stage enamel formation in Lhx6 null mice. Amelogenin and ameloblastin expression is reduced and/or delayed in the Lhx6 null mice potentially resulting from defects in dentin deposition and ameloblast differentiation. Our results demonstrate that Lhx6 regulates cell proliferation in the cervical loop and promotes cell differentiation in the anterior region of the incisor. We demonstrate new molecular mechanisms for Lhx6 and an interaction with PITX2 for normal craniofacial and tooth development.
[Show abstract][Hide abstract] ABSTRACT: Amelogenin, the major protein of forming dental enamel, plays a crucial role in the biomineralization of this tissue. Amelogenin is soluble at low pH and self-assembles to form higher order structures at physiological pH. To understand the mechanisms of its assembly and interactions with calcium phosphate mineral, we conducted FTIR spectroscopy (FTIRS) studies of pH-triggered assembly of recombinant porcine amelogenin rP172 and its interactions with mature hydroxyapatite and apatitic mineral formed in situ. Analysis of our data indicated that rP172 at pH 3.0 exists in an unfolded disordered state, while increases in pH led to structural ordering, manifested by increases in intra- and intermolecular β-sheet structures and a decrease in random coil and β-turns. Amelogenin assembled at pH 7.2 was also found to contain large portions of extended intramolecular β-sheet and PPII. These FTIRS findings are consistent with those previously obtained with other techniques, thus verifying the validity of our experimental approach. Interestingly, interactions with mineral led to a reduction in protein structural organization. The findings obtained show that amelogenin has intrinsic structural flexibility to accommodate interactions with both forming and mature calcium phosphate mineral phases, providing new insights into the potential importance of amelogenin-mineral interactions in enamel biomineralization.
Journal of dental research 08/2012; 91(10):967-72. DOI:10.1177/0022034512457371 · 4.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Growth factors such as platelet derived growth factor (PDGF) have significantly enhanced periodontal therapy outcomes but with a high degree of variability, mostly due to lack of their continual supply for a required period of time. One method to overcome this barrier is gene therapy and the aim of this in vitro study is to evaluate PDGF-B gene delivery in fibroblasts using nano-sized calcium phosphate particles (NCaPP) as vectors. Methods: NCaPP incorporating green fluorescent protein, GFP (NCaPP-GFP) and PDGF-B (NCaPP-PDGF-B) plasmids were synthesized using an established precipitation system and characterized using transmission electron microscopy and 1.2% agarose gel electrophoresis. Biocompatibility and transfection of the nanoplexes in fibroblasts were evaluated using cytotoxicity assay and florescence microscopy, respectively. Polymerase chain reaction (PCR) and enzyme linked Immunosorbent assay (ELISA) were performed to evaluate PDGF-B transfection after different time points of treatments and the functionality of PDGF-B transfection was evaluated using the cell proliferation assay. Results: Synthesized NCaPP nanoplexes incorporating the genes of GFP and PDGF-B were spherical in shape and measured about 30 to 50 nm in diameter. Gel electrophoresis confirmed DNA incorporation and stability within the nanoplexes and MTS assay demonstrated their biocompatibility in fibroblasts. In vitro transfection studies revealed a higher and longer lasting transfection after NCaPP-PDGF-B treatment, which lasted up to 96 hours. Significantly enhanced fibroblast proliferation observed in NCaPP-PDGF-B treated cells confirmed the functionality of these nanoplexes. Conclusion: NCaPP demonstrated higher levels of biocompatibility and efficiently transfected PDGF plasmids into fibroblasts under described in vitro conditions.
Journal of Periodontology 03/2012; 84(1). DOI:10.1902/jop.2012.120012 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amelogenin is essential for proper enamel formation. The present in vitro study extends our previous work at low (10 mM) ionic strength (IS) by examining the effect of amelogenin on mineralization under higher (162 mM) IS conditions found in developing enamel. Full-length phosphorylated (P173) and non-phosphorylated (rP172) amelogenins were examined, along with P148 and rP147 that lack the hydrophilic C-terminus. Calcium phosphate formation was assessed by pH change, while the minerals formed were characterized using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. Amelogenin self-assembly was also studied using dynamic light scattering and TEM. The results indicate that IS does not influence the effects of rP147, rP172, and P173 on mineralization. However, in contrast to the findings for low IS, where both P173 and P148 stabilize initially formed amorphous calcium phosphate (ACP) nanoparticles for >1 d, elongated hydroxyapatite crystals were observed after 24 h using P148 at high IS, unlike that seen with P173. Differences in self-assembly help explain these findings, which suggest that P173 and P148 may play different roles in regulating enamel mineral formation. The present data support the notion that proteolytic processing of P173 is required in vivo to induce the transformation of initial ACP phases to apatitic enamel crystals.
European Journal Of Oral Sciences 12/2011; 119 Suppl 1(suppl. 1):103-11. DOI:10.1111/j.1600-0722.2011.00911.x · 1.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly. Furthermore, our results indicate that interactions between amelogenin hydrophilic C-terminal telopeptides are essential for oligomer formation and for subsequent steps of hierarchical self-assembly. We further show that amelogenin assemblies stabilize mineral prenucleation clusters and guide their arrangement into linear chains that organize as parallel arrays. The prenucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites, the hallmark structural organization of the forming enamel at the nanoscale. These findings provide unique insight into the regulation of biological mineralization by specialized macromolecules and an inspiration for bottom-up strategies for the materials design.
Proceedings of the National Academy of Sciences 08/2011; 108(34):14097-102. DOI:10.1073/pnas.1106228108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amelogenin's capacity to regulate enamel formation is related to its conserved N- and C-terminal domains, its ability to self-assemble, and its ability to stabilize amorphous calcium phosphate (ACP) - a capacity enhanced by amelogenin phosphorylation. This in vitro study provides further insight into amelogenin function, using variations of the Leucine-Rich Amelogenin Peptide (LRAP), an alternative splice product comprised solely of amelogenin's N- and C-terminal domains. Peptide self-assembly was studied by dynamic light-scattering and transmission electron microscopy (TEM). TEM, selected area electron diffraction, and Fourier transform-infrared spectroscopy were also used to determine the effect of phosphorylated and non-phosphorylated LRAP on calcium phosphate formation. Results show that phosphorylated and non-phosphorylated LRAP can self-assemble into chain-like structures in a fashion dependent on the C-terminal domain. Notably, this capacity was enhanced by added calcium and to a much greater degree for phosphorylated LRAP. Furthermore, phosphorylated LRAP was found to stabilize ACP and prevent its transformation to hydroxyapatite (HA), while aligned HA crystals formed in the presence of non-phosphorylated LRAP. The N- and C-terminal amelogenin domains in non-phosphorylated LRAP are, therefore, sufficient to guide ACP transformation into ordered bundles of apatite crystals, making LRAP an excellent candidate for biomimetic approaches for enamel regeneration.
Journal of dental research 06/2011; 90(9):1091-7. DOI:10.1177/0022034511411301 · 4.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryogenic transmission electron microscopy (cryo-EM) was used to explore the self-assembly of recombinant murine amelogenin (rM179) in vitro. Our cryo-EM data showed that amelogenin self-assembly is a strongly pH-dependent process. At pH 4.4 the main fraction of the protein exists in a monomeric form, although some peculiar structures consisting of chains of monomers were also observed. At pH 5.8 large nanospheres comprising ring-like structures ~50 nm in diameter were the most abundant particle class. Similarly, at pH 8.0 amelogenins self-assembled into ring-like oligomers of different sizes, which subsequently assembled into nanospheres 15-20 nm in diameter. Furthermore, at pH 7.2, which is close to a physiological pH, branched chains of nanospheres were observed. Our results show that amelogenin assembly is a multistep hierarchical process and provides new insight into the control of enamel mineralization.
[Show abstract][Hide abstract] ABSTRACT: N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (-P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(-P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.
[Show abstract][Hide abstract] ABSTRACT: Introduction of growth factors such as PDGF into clinical practice has improved the prognosis of complex clinical scenarios but with a high degree of variability. This is, in part, is due to a lack of a continual supply of these proteins for a prolonged period of time. One method to overcome this drawback is gene therapy. Objective: To develop a nano calcium phosphate based gene delivery system that can be employed to deliver genes of interest into fibroblasts in the periodontal defect. The transfected cells will subsequently act as local protein synthesizing machinery. Methods: Calcium phosphate nano particles encapsulating the DNA of interest (CaPDNA) were synthesized using an established protocol. Briefly, a solution containing calcium and DNA was added in a drop-wise manner into phosphate solution, under gentle mixing. The particles synthesized were then characterized for size, shape and chemical composition using dynamic light scattering (DLS), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR). DNA stability within the nano particles and the DNA encapsulation efficiency were determined using 1.2 % agarose gel electrophoresis and spectroscopic (OD 260nm) techniques, respectively. In vitro cell culture was utilized to determine the biocompatibility and transfection efficiency of the synthesized nano particles using murine fibroblasts (NIH-3T3). MTS assay and florescence microscopy were employed to determine cytotoxicity and transfection efficiency, respectively. Results: The nano particles were found to be spherical in shape with diameters of about 30 to 50 nm. The particles were comprised of both amorphous as well as hydroxyapatite phases. DNA encapsulation efficiency was found to be around 85% (data not shown) and agarose gel electrophoresis confirmed DNA stability after encapsulation within the nano particles. The vectors were highly biocompatible after 6 hours of treatment (MTS assay). The in vitro transfection study demonstrated high levels of transfection at 48 hours of treatment with murine fibroblasts. Conclusion: Calcium phosphate nano particles can potentially serve as a good vehicle to deliver target genes to fibroblasts for periodontal regenerative purposes.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs are known to regulate gene function in many tissues and organs and in this study we investigate the overall functions of microRNAs in tooth development. Objectives: We have identified discrete sets of mircoRNAs expressed in molars and incisors as well as epithelium and mesenchyme. Specific set of microRNAs play a role epithelial cell proliferation, differentiation and amelogenesis. Methods: We use transient transfections, real-time PCR, mutant mice, chromatin immunoprecipitation (ChIP) assays, electrophoretic mobility shift assays, immunochemical and biochemical assays. Results: Conditional knockout (cKO) of Dicer1 (mature microRNAs) in the dental epithelium using the Pitx2-Cre mouse results in multiple and branched enamel-free incisors and cusp-less molars. Analyses of differentiating dental epithelial markers reveal a defect in ameloblast differentiation. Conversely, the cervical loop (stem cell niche) is expanded in Dicer1 cKO. Epithelial microRNAs control dental stem cell differentiation demonstrating a unique role for microRNAs in regulating in vivo dental stem cell biology. Noggin, a potent BMP inhibitor is up-regulated in the Pitx2cre-Dicer1 knockout mouse, which prevents BMP signaling and cell differentiation. We found miRNA-200c can target Noggin in vitro, which may responsible for ameloblast cell differentiation defects. We also made miRNA-200c transgenic mice that overexpress miRNA-200c. Analysis of this mouse should shed on miRNA-200c's function during tooth development. Conclusions: These results demonstrate a critical role for microRNAs in regulating tooth and craniofacial development and a mechanism involving miRNA-200c regulation of Noggin and BMP signaling. Support for this research was provided from grant DE13941 from the National Institute of Dental and Craniofacial Research.
[Show abstract][Hide abstract] ABSTRACT: The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed.