Materials Science and Engineering C 32 (2012) 858–861
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Materials Science and Engineering C
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Modulation of enamel matrix proteins on the formation and nano-assembly ofhydroxyapatite in vitro
Hong Li a,b,⁎, Weiya Huang c,d, Yuanming Zhang c, Bo Xue a, Xuejun Wen b
a Department of Materials Science and Engineering, Jinan University, Guangzhou, Guangdong 510630, Chinab Department of Bioengineering, Clemson University, Charleston, SC 29425, USAc Department of Chemistry, Jinan University, Guangzhou, Guangdong 510630, Chinad Department of Materials Science and Engineering, Taizhou, Taizhou University, Zhejiang 317000, China
⁎ Corresponding author at: Department of MaterialsUniversity, Guangzhou, Guangdong 510630, China. Tel.:20 85226663.
Natural enamel has a hierarchically nanoassembled architecture that is regulated by enamel matrix proteins(EMPs) during the formation of enamel crystals. To understand the role of EMPs on enamel mineralization,calcium phosphate (CaP) growth experiments in both the presence and absence of native rat EMPs in a singlediffusion system were conducted. The morphology and organization of formed CaP crystals were examined byX-RayDiffraction (XRD),High-Resolution TransmissionMicroscopy (HRTEM) and Selected Area ElectronDiffrac-tion (SAED). In the system containing the EMPs, hydroxyapatite (HAP) with hierarchical lamellar nanostructurecan be formed and the aligned HAP assembly tightly bundled by 3–4 rod-like nanocrystals like an enamel prism.However, in the absence of EMPs, only a sheet-like structure of octacalcium phosphate (OCP) phase waspresented. EMPs promote HAP formation and inhibit the growth of OCP on the (010) plane. It is discussed thatthe organized Amelogenin/Amorphous Calcium Phosphate might be the precursor to the bundled HAP crystalprism. The study benefits the understanding of biomineralization of tooth enamel.
Maturemammalian tooth enamel is the hardest, most highlyminer-alized tissue in the vertebrate body. Although enamel has the samechemical composition as its prototypematerials such as hydroxyapatite(HAP), tooth enamel crystal shows some unique characteristics:extremely elongated in the c-axial direction, high aspect ratio andpacked in parallel arrays to form enamel prisms which constitutetooth enamel [1]. These micro-architectures are organized from bio-mineralization, indicating that a significant aspect of biological controlovermineral formation is through protein/inorganic recognition and in-teraction. It has been suggested that the predominant enamel matrixproteins (EMPs), amelogenin (Amel), self-assembles to form supramo-lecular nanospheres that facilitate the nano-assembly organization ofinorganic structures in developing enamel crystals [1–4]. However,the process of tooth enamel biomineralization has not yet been directlyidentified, especially for the nano-assembled structure of tooth enamel.Extensive experimental evidences support the hypothesis that enamelapatite formation involves octacalcium phosphate (OCP) as a precursorphase [5–10]. The overgrowth of apatite occurs on the (100) faces of thetemplate OCP crystals with the blocking of the side face by Amel
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nanospheres, resulting in an increase in crystal thickness in the [100]direction, with the eventual formation of tightly assembled prisms[7,8,11]. However, no evidence has shown the direct transformationfrom OCP to HAP with a nano-assembled structure. In recent studies,hierarchically organized apatite microstructures were achieved in thepresence of Amel at the initial crystallization of the apatite [12,13]. Ithas been hypothesized that the presence of Amel promotes the assem-bly of Amel/Amorphous Calcium Phosphate (ACP) nanosphere chains,and further induces nanorod HAP formation. Due to the difference inthe experimental systems in the study of calciumphosphate crystalliza-tion, the modulation mechanism of EMPs or Amel on composition andmorphology of calcium phosphate crystals is still unclear, especially inrelation to howwell it can control the formation of specifically orientednano-assembled prisms.
In the study, we focused on the apatite crystallization using theextracted rat EMPs in an agarose gel system. The gel network is an ex-cellent system for studying crystallization because (a) protein is incor-porated into the gel at room temperature and remains stable duringthe experiment; (b) the local concentration for crystallization is readilyachievable; (c) the deposits are easily harvested from the mediumwithout agarose contamination [14–17]. In the gel system, the physico-chemical nature more realistically mimics a mineralized tissue matrixenvironment, and EMPs control apatite crystallization on both composi-tion and morphology. A tightly bundled hierarchically nano-assembledHAP crystal was similar to natural enamel obtained in the study. The re-sults verified EMPs assembly calcium phosphate into nano-assembled
Fig. 1. XRD patterns of the products grew in the system: (a) in the absence and (b) inthe presence of EMPs. The standard patterns of OCP and HAP were taken according toPDF 44-0778 and 09-0432 respectively.
Fig. 2. SEM images of crystals grew in the absence (a) andpresence (b) of EMPs, respectively.Note that the ribbon-like crystals in (b) are narrower and longer than those in (a).
859H. Li et al. / Materials Science and Engineering C 32 (2012) 858–861
prism structure in vitro, and also suggested that EMPs induced theformation of ordered ACP in a hierarchical structure to evolve intotooth enamel HAP.
2. Materials and methods
2.1. Selective extraction of EMPs from rat molars
25 rats were obtained from Animal Centre (Animal Centre of SunYat-Sen University, Guangzhou, China), and all of whichwere sacrificedusing the immortal method approved by the Institutional Animal Careand Use Committee of Sun Yat-Sen University. The rats were intrave-nously anesthetized by 2% pentobarbital sodium (30 mg/kg). Rat enam-el matrix proteins (EMPs) were extracted using an establisheddissociative extraction [18]. The protein solution was lyophilized andstored at−80 °C, extracted and examined by SDS-gel shown in SupportInformation 1(SI. 1). The composition of EMPs was Amel-rich extractand estimated to mainly contain 95% of R180, 27 kDa.
2.2. Growth of apatite crystals
Apatite crystals were synthesized by the controlled chemical reac-tion in an agarose gel with or without EMPs under both physiologicalpH and temperature environment. The processing is described in detailin SI. 2. This gel system, known as a single diffusion system [15], contin-uously operated for 3 days, and a layer of white product was seen at theinterface of Ca2+ solution and PO4
3− gel both with and without EMPs.Prior to being coated with platinum for SEM observation, the productswere carefully removed from the interface and washed with warmdeionized water 3 times without shaking. For XRD and TEM observa-tions, the samples were heated to 50 °C with deionized water, centri-fuged while warm, and abandoned the upper solution. Further, theproducts were cleansed of agarose by washing with warm water andthen centrifuged several times until the upper solution was clear.
2.3. Characterization
XRDmeasurements (XRD, MSALXD-2)were taken of CuKα at 40 kVand 20 mA. A thin platinum film was coated on the samples for SEMobservation (via a Philips XL-30ESEM operated at 30 kV). The crystalswere studied using TEM (with a JEM-200CX microscope) in whichselected area electron diffraction (SAED) patterns were also recorded.The samples for TEM observation were dispersed by ultrasonic treat-ment in ethanol medium before being placed onto Cu grids that werecovered previously with carbon films.
3. Results
Fig. 1 shows XRD patterns of the products obtained in the single-diffusion system with the absence and presence of EMPs. Regardingthe sample with the absence of Amel (Fig. 1a), the sharp (100) reflec-tion at 2θ=4.7° was a characteristic of OCP phase, indicating that theproduct was mainly OCP, together with the other characteristic OCPpeaks of (200) and (010). Further, a series of sharp peaks between2θ=31° and 34° also indicates that the main phase is OCP. No obvious(100) reflection of HAP at a degree of 2θ=10.8° appears, whichwould be an evidence of either no or less HAP formation in the gel sys-temwith the absence of EMPs. Characteristic XRD peaks at 2θ=26° and32° attributed to HAP were observed in the product with the presenceof EMPs (Fig. 1b), indicating that HAP was the major precipitated crys-tals. The characteristic reflection of OCP was still observed at a low de-gree of 2θ=4.7° at Fig. 1b with weak intensity; hence, the productwith the presence of EMPs was a mixture of HAP and OCP.
Fig. 2a shows SEM images of crystals obtained from the systemwith the absence of EMPs. These sheet-like crystals, 5 μm in widthand 25–30 μm in length with a blade-edge end, were broken in TEM
images due to the ultrasonic treatment at sample preparation stage,as shown in Fig. 3. The SAED insertion in Fig. 3 shows that thesesheet-like shape crystals are OCP with wide (100) faces, which aresimilar to those reported previously [19,20]. No other particles wereobserved. The results show that the product in the system with theabsence of EMPs was mainly OCP. When the size of the sheet-likeOCP crystals is compared to the ribbon-like crystals with EMPs(Fig. 2b), it was observed that EMPs decreased the width of the OCPcrystals remarkably, as observed previously with Amel [5,21].
In the sample with EMPs, the ribbon-like crystals grew in a meanwidth of approximately 510±20 nm(n=15)with a continuous exten-sion, together with fine and needle-like crystals (Fig. 2b). These needle-shaped crystals appear to form aggregations with a random dispersionas shown in Fig. 2b. Fig. 4 details the various morphologies of the crys-tals precipitated in the reactor systemwith the presence of EMPs usingTEM observation. It is shown that plate-like crystals in a very thin struc-ture with typical diffraction pattern of OCP with a wide (100) face areattributed to the ribbon-like crystals in Fig. 2b. A rod-like crystal witha large thickness (indicated by the asterisk “*”) in Fig. 4a, which isapproximately 30 nm in depth, 80 nm in width and 450 nm in lengthis shown in Fig. 4d for further details. The “*” crystal has a 2–3 multiplelayer structure, indicating that it has been assembled by at least twolamellar nanocrystals along a defined plane. The inserted SAED(Fig. 4c) shows that the assembled crystal with a pattern of HAP diffrac-tion. This implies that a rod-like hierarchical HAP crystal might beoriginated from the nano-assembled organization of sheet-like orlamellar crystals.
Fig. 3. TEM image of the crystals grew in the system with the presence of EMPs and itsinserted ED taken from to the [110] zone axis.
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An assembled crystal prism in Fig. 5 with appreciable nanocrystalsaligned pack also was observed in the sample with the presence ofEMPs, inwhichHRTEMwas used to observe the alignment of nanocrys-tals along the preferentially oriented c-axes of HAP (Fig. 5a and c). Theassembled crystal prism in a width of 80 nmwasmost likely an assem-bly of 3–4 rod-like crystals in awidth of 30–50 nm. This structure is sim-ilar to the apatite structure of a natural tooth enamel prism, which has atightly parallel packed configuration of elongated nanocrystals. Theassembled structure of apatite crystallization controlled by Amel waspredicted in earlier work [22]. Fig. 5b also shows that the rod-like crys-tals with quasi-hexagonal tips seem to be organized by lamellar crystalsin a defined crystal plane. The organization also coincides with thestructure of which the single nanocrystal “*” is shown in Fig. 4d.
4. Discussion
The purpose of our experiments is to study the apatite crystallizationmodulation and nano-assembled organization depending EMPs mainlycontaining Amel. Amel self-assembly nanosphere is believed to be atemplate of enamel formation [3,22]. However, a further assembly ofcrystalline structure analogous to that of tooth enamel was lessobserved. Here the assembled crystal in our study verifies in somedegree that EMPs modulate CaP nanocrystals to convert into final
Fig. 4. TEM images of the crystals formed in the systemwith presence of EMPs. (a)MorphologieFig. 2b). (b) The SAED pattern of the circle area in Fig. 4a corresponds to the (100) plane of OCPcrystal assembled by 2–3 lamellar nanocrystals along a defined plane.
oriented bundled HAP crystals (see Fig. 5b). It was presumed that OCPwould fulfill the precursor of enamel apatite, because it crystallizesinto a sheet-like or ribbon-like morphology, which is identical to thework findings in our crystal sample (Fig. 2a). Also, because OCP nucle-ates more rapidly than HAP under physiological medium [23], it wasthe main crystals in most biomimetic systems [5,10,24]. Amel oftenblocks the OCP growth by absorbing Amel nanospheres in the sideface, and decreases in the width of the sheet-like OCP crystals, asFig. 2 shown. This blocking also actively induces the [100] growth ofOCP, and increases the thickness of OCP crystals, resulting in rod-likeapatite in the same crystal orientation according to the reports[21,25]. However but no promotion of HAP hierarchical assembly byAmel was reported. It has also been suggested that amorphous mineralorganization using Amel matrix control occurs during the early stage ofenamel formation prior to crystallization [8,9,12,26]. However, the finalelongated and bundled HAP crystals prism still cannot be acquired invitro. Recent investigations strongly suggested that themineralizedma-trix might be assembled into a mineral chain through an amorphousprecursor phase [12,14,26]. During early enamel formation, the nano-sphere chain of Amel/ACP was organized into a parallel structure, andsubsequently grew according Ostwald's ripening process to form hier-archical organization [12].
An aligned packedHAP structurewith 3–4 rod-like nanocrystalswasobtained in our system with EMPs (mainly containing Amel) in Fig. 5,while the rod-like nanocrystal also organized by the very thin lamellarcrystals in Fig. 4d. It is difficult to know from the morphologies of ourproducts whether the oriented growth of OCP or the organized packof Amel/ACP facilitated HAP crystal organization. We propose that or-dered Amel/ACP chains were the precursor of aligned packed HAP forthe size of Amel/ACP precursor coincides with that of the assembledHAP crystal. OCP usually shows its micrometer-sized crystals, however,Atomic ForceMicroscope images of apatite crystals from developing ratenamel showed a nanoparticle chain with a width of 80 nm [26]. Thenanospheres of rp172 Amel/ACP also have a uniform diameter of ap-proximately 60 nmas observed in a CaP system [27]; and the precipitat-ed ACP also consists of aggregates of primary nuclei in 30–80 nmnanoparticles in the previous reports [28,29]. These configurations areconsistent with our results. The observed nanocrystal with dimensionof 30–80 nm bundled together. We predicted that the self-assembly ofAmel would initiate in a solution and also finished before it wasadded to the gel [15], because the rearrangement of macromoleculesin vitro can either be prevented or retarded in a gel medium. However,the assembled Amel may be affected by the PO4
3− inorganic ions, caus-ing the formation of Amel-PO4
3− ordered clusters. When Ca2+ reachedvia diffusion through the gel medium, the Amel/ACP deposited on theAmel-PO4
3− ordered clusters. We could not demonstrate the existenceof Amel-PO4
3− due to the limitations of existing experimental
s of the obtained crystals (The sheet-like crystals in Fig. 4a are related to the ribbon-like in. (c) The SAED pattern of the asterisk “*” crystal in Fig. 4a corresponds to HAP. (d) The “*”
Fig. 5. TEM images of an assembled crystal in the system with EMPs. (a) and (c) are the HRTEM of the crystal in 2 rectangle areas in (b). The measured lattice spacing of approx-imately 0.346 nm corresponds to the (002) HAP lattice plane.
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approaches for monitoring in situ particle evolution in a gel system.However, Busch et al. hypothesized that ions (e.g. Ca2+ and PO4
3−)strongly affected macromolecular assemblies and reported parallelHAP crystals in a gelatin gel system [30]. The organized nanosphereAmel/ACP would provided an opportunity for HAP crystallization in aminimum free energy state [12,31], and an aligned HAP assemblywith 3–4 nanocrystals in an ordered nanostructure was formed in ourreactor system.
5. Conclusion
CaP crystals growth experiment in both the presence and absenceof native rat Amel-rich matrix proteins in a single diffusion systemwas conducted under the physiologic conditions of a pH of 7.25 and37 °C. Rod-like HAP nanocrystals assembled by ordered lamellarnanocrystals were formed in the presence of EMPs, and aligned HAPassembly tightly bundled with the 3–4 rod-like HAP nanocrystals ina hierarchical structure. EMPs promoted HAP formation and inhibitedthe growth of OCP with a face of the (010) plane. However, withoutthe presence of EMPs, sheet-like structure of OCP phase was mainlypresented. These results demonstrate that the EMPs assemble calci-um phosphate to evolve into a bundled prism structure, permittingevolution into hierarchical enamel HAP.
Acknowledgment
This research was supported by the grant 30870612 and 31070852from the National Science Foundation of China
The authors thank Neil Mullen for language system.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.1016/j.msec.2012.02.003.
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