Polyaspartic Acid Concentration Controls the Rate of Calcium

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Polyaspartic acid concentration controls the rate of calcium phosphate nanorod formation in high concentration systems Daniel V. Krogstad, Dongbo Wang, and Sheng Lin-Gibson Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00772 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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Polyaspartic acid concentration controls the rate of calcium phosphate nanorod formation in high concentration systems Daniel V. Krogstad,† Dongbo Wang, Sheng Lin-Gibson* Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD Abstract Polyelectrolytes are known to greatly affect calcium phosphate (CaP) mineralization. The reaction kinetics as well as the CaP phase, morphology and aggregation state depend on the relative concentrations of the polyelectrolyte and the inorganic ions in a complex, non-linear manner. This study examines the structural evolution and kinetics of polyaspartic acid (pAsp) directed CaP mineralization at high concentrations of polyelectrolytes, calcium, and total phosphate (19 to 30 mg/mL pAsp, 50 to 100 mM Ca2+, Ca/P = 2). Using a novel combination of characterization techniques including cryogenic transmission electron microscopy (cryo-TEM), spectrophotometry, X-ray total scattering pair distribution function analysis, and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), it was determined that the CaP mineralization occurred over four transition steps. The steps include: the formation of aggregates of pAsp stabilized CaP spherical nanoparticles (sNP), crystallization of sNP, oriented attachment of the sNP into nanorods, and further crystallization of the nanorods. The intermediate aggregate sizes and the reaction kinetics were found to be highly polymer concentration dependent while

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the sizes of the particles were not concentration dependent. This study demonstrates the complex role of pAsp in controlling the mechanism as well as the kinetics of CaP mineralization. Keywords Calcium phosphate, polyaspartic acid, biomineralization, oriented attachment Introduction Bone possesses exceptional materials properties and therefore has been extensively studied. The hierarchical assemblage of calcium phosphate (CaP) platelets and collagen convey to bone many exceptional material properties. A diverse class of charged proteins known as noncollagenous proteins (NCP)1,2 directs the hierarchical assembly of CaP platelets into the collagen matrix. The roles of NCP are numerous and complex; key functions of NCP include stabilizing CaP precursor phases and mediating the CaP mineralization process.1,2 Recent in vitro studies on biomimetic mineralized collagen have shown that it is possible to produce a structure very similar to the mineralized collagen in bone using polyaspartic acid (pAsp) as a NCP mimic.1 3,4 Without pAsp, collagen fibers do not properly mineralize; however, the exact mechanism by which pAsp controls the mineralization remains elusive. To better understand the role of pAsp, CaP mineralization in the absence of collagen has been extensively studied. These studies showed the mechanism of CaP mineralization and the shape/structure of the particles are highly sensitive to the concentration of calcium (Ca2+), total phosphate (Pi), and pAsp as well as other reaction conditions (i.e., temperature, pH, ionic strength).3-11 Our previous work examined the effects of the overall and relative concentrations of Ca2+, Pi and pAsp in the mineralization process. In one study, CaP spherical nanoparticles (sNP) of 500 nm) were prominent; whereas in both samples with 30 mg/mL pAsp, significantly smaller (< 100 nm) aggregates were more pervasive. These results suggest pAsp is important in stabilizing the sNP aggregates as well as the individual sNP, likely through electrostatic repulsion between sNP.


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The effects of pAsp and ion (Ca2+, Pi) concentrations on CaP particle formation were studied in situ by monitoring the optical density (OD) as a function of time using a spectrophotometer (Figure 3, Figure S1). In this system, changes in OD are related to changes in particle size and aggregation state (resulting in changes in scattered intensity), and in some cases, due to sedimentation of the aggregates. Generally, the OD decreases with a decrease in the aggregate size and increases with increasing particle size (sNP to rod). The OD profiles for the three samples that had 50 mM calcium and varying pAsp concentration are shown in Figure 3a. Consistent with our previous results, aggregates of sNP formed within 3 min after mixing,8 the time at which the first data point could be collected. For the 24 and 30 mg/mL pAsp concentrations, there was an overall increase in the OD with time corresponding to the sNP to rod transition. For the 19 mg/mL sample, the initial OD was significantly higher than the final OD. The initial high OD corresponds to the presence of very large aggregates, which is consistent with the initial cryo-TEM images (Figure 2a). Over time, the OD of the 19 mg/mL sample decreased and eventually converged to a similar value to the samples with higher pAsp concentrations. Cryo-TEM taken at intermediate time points (Figure 3d) showed that the aggregates dissociated with time. The local minimum in the OD observed at a reaction time of ≈ 4 h was a result of the simultaneous dissociation of the aggregates and the oriented attachment of spheres into nanorods. The agreement between OD data and cryo-TEM results provided us with confidence that OD measurements can be used as an in situ approach to continuously monitor the structural evolution of the CaP mineralization for the compositions studied.


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Figure 3: Plots of the optical density (OD) of the samples as measured with a spectrophotometer at a wavelength of 650 nm. a) Light transmission was monitored every minute over 24 hours for all three of the 50 mM Ca2+ samples to show changes in the OD of the reactions. b) The beginning (3 min, open symbols) and end (24 hrs, closed symbols) points for all nine samples are shown. The data indicates that the OD decreases with increasing pAsp concentration or decreased ion concentration. The lines were drawn for clarity to show the trends. The cryo-TEM images of the 19 mg/mL 50 mM Ca2+ at c) 15 min, d) 4 hrs, and e) 24 hrs show the presence of large aggregates initially and then the subsequent disassociation of the aggregates and the formation of nanorods.


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It should be noted that for all of the 75 mM Ca2+ samples, some sedimentation of the aggregates was observed. The results of this can be seen as an initial drop in the OD for the 19 mg/mL sample in Figure S1. For the 24 mg/mL and 30 mg/mL samples, the cuvettes were periodically shaken in order to prevent the sedimentation. No sedimentation was observed for the 50 mM Ca2+ and 100 mM Ca2+ solutions. The 50 mM Ca2+ samples were highly soluble, preventing sedimentation. The 100 mM Ca2+ solutions were concentrated enough that the sedimentation did not occur. However, the 19 mg/mL pAsp 100 mM Ca2+ sample did have some initial precipitation that crashed out of solution before the first OD measurements could be made. Figure 3b shows the initial (3 min, dashed) and final (24 h, solid) ODs for all compositions studied. Generally, the initial OD increased when the Ca2+ concentration increased or when the pAsp concentration decreased (i.e., decrease in pAsp/Ca2+ ratio) indicating an increased size of the initial sNP aggregates. Additionally, most of the compositions reached comparable final ODs, consistent with the formation of dispersed rods. The only exception was the 19 mg/mL pAsp, 100 mM Ca2+ sample, where some precipitates were observed. Therefore, this sample may be close to the pAsp concentration in which the CaP formation can no longer be stabilized in suspension. Taken together, these results show that within this concentration regime, the initial aggregation size is dependent on both the pAsp and the ion (Ca2+, Pi) concentration. The particle size, on the other hand, is independent of both the pAsp and the ion (Ca2+, Pi) concentration. The atomic structure (phase) evolution was further investigated via in situ synchrotron X-ray total scattering pair distribution function (PDF) analysis. The atomic PDF, or G(r), plots the distribution of atom-to-atom pairs as a function of distance in a material and can be used to understand the atomic structure of a material. PDF data from the necessary reference materials (NIST SRM 2910b Hydroxyapatite, pAsp in HBS, pAsp and Ca2+ in HBS, pASp and Pi in HBS)


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are shown in Figure S2. A comparison of the PDF results of hydroxyapatite and pAsp in HBS indicated that features below 7 Å were from a combination of the hydroxyapatite and pAsp; while features above 7 Å were due entirely to hydroxyapatite since the flexibility of the polymers prevented a significant contribution to measured medium range order. In particular, the peak at 9 Å was identified as a potential peak for monitoring the presence of the apatite structure since it is a prominent peak and is not found in other CaP minerals (e.g., ACP, DCPA and DCPD). From our simulations using PDFgui, this distance corresponded to two calcium ions in adjacent unit cells, so it most likely represented medium range ordering within the mineral (Figure S3). In situ PDF data for the 30 mg/mL pAsp 50 mM Ca2+ sample collected at several time points (Figure 4) showed that the 9 Å peak did not change from 6 h to 24 h, indicating that the sample was wellordered by 6 h. In contrast, for the measurement taken at 1 h, no peak was present at 9 Å and corresponded to less ordered CaP phase. It should be noted that fast changes in the structural transformation could not be monitored by in situ PDF as each solution measurement took 30 min to collect. On the other hand, PDF data provides conclusive evidence for the transition from a less ordered to a more ordered structure over time. Additional PDF curves for the remaining eight samples are shown in Figure S4. To our knowledge, this is the first use of PDF to study in situ mineralization at such low concentrations (< 0.4 % by mass mineral) and may serve as a powerful method to study crystallization by particle attachment.17


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Figure 4: Pair distribution function data for the 30 mg/mL pAsp 50 mM Ca2+ sample taken at 1, 6, 12 and 24 hours after mixing. The peak at roughly 9 Å is indicative of apatite formation and medium range order spanning two unit cells. It can be seen that this peak is not present at 1 hour, but reaches the full height by 6 hours. The inset shows the expanded region in the gray square.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was then used to obtain more temporally resolved information about the atomic structure (phase) evolution (Figure 5). Spectra were collected every 5 min. The ATR-FTIR spectra show peaks between 1750 cm-1 and 1300 cm-1 assigned to pAsp, generally the amide modes,18 and peaks between 1150 cm-1 and 950 cm-1 assigned to phosphate.19-22 The spectral information associated with phosphate can be used to classify atomic ordering of the CaP phase. Crystalline phases and


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disordered phases can be easily differentiated.21-23 To identify apatite, multiple reports have reported the ν3 PO43- antisymmetric stretch in the 1030 cm-1 to 1033 cm-1 range.19,21,22 In octacalcium phosphate (OCP), ν3 PO43- antisymmetric stretch occurs at slightly lower wavenumbers (1021 cm-1 to 1028 cm-1).19,21,22 Results on amorphous calcium phosphate (ACP) are more varied; however, a broad peak has been observed at 1052 cm-1 22 but can shift up to 1085 cm-1 depending on the phosphate speciation (HPO42- or PO43-).24, 25 Solution H2PO4- and HPO42- peaks have also been reported at 1077 cm-1 and 1080 cm-1, respectively.19,21 In our analysis, the peaks at 1024 cm-1 and 1032 cm-1 were considered ordered peaks whereas the broad peak centered at 1076 cm-1 was considered disordered; this broad peak likely contained contributions from the H2PO4- and HPO42- in solution and in ACP. A similar approach was previously utilized to study the ACP to apatite solution mediated transformation process.23

Figure 5: ATR-FTIR absorbance data for the 30 mg/mL pAsp 50 mM Ca2+ sample collected every hour for 12 hours. The peaks in the 1300-1700 cm-1 range are assigned to pAsp and the peaks in the


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950-1150 cm-1 range are assigned to phosphate. The samples have been normalized to the pAsp peak at 1400 cm-1. The inset shows the change over time in absorbance values for the 1024 cm-1, 1032 cm-1 and 1076 cm-1 peaks. Data was taken every 5 minutes for 12 hours. The results show that the ordered peaks, 1024 cm-1 and 1032 cm-1, increase with respect to 1076 cm-1 over the entire range.

ATR-FTIR spectra for the 30 mg/mL pAsp, 50 mM Ca2+ sample collected as a function of time over 12 h is shown in Figure 5. The data was normalized to the pAsp peak (1398 cm-1), because we were most interested in changes in peak intensities related to phosphate (between 1150 cm-1 and 950 cm-1).19-22 The absorbance for two ordered phosphate peaks (1024 cm-1 and 1032 cm-1, respectively) and one disordered phosphate peak (1076 cm-1) are shown in the inset of Figure 5. In secretory stage enamel, phosphates near the surface have a significant feature in FTIR between 1100 cm-1 and 1070 cm-1; however near the center of the enamel, the more mature ordered structure, this peak was almost nonexistent.20 Interestingly, the ATR-FTIR spectra at the initial time points are more comparable to the young enamel at the surface while the later time points are more comparable to the mature enamel at the center indicating that the mechanism observed in this analysis may be very similar to biological CaP formation. The change in ordering of the mineral phase was monitored over time via the ordered to disordered phosphate peak ratio (1032 cm-1/1076 cm-1) (Figure 6, Figure S5). Two rapid changes in the 1032 cm-1/1076 cm-1 peak ratio were observed from ATR-FTIR. We attribute these rapid changes to phase transitions or increase of ordered phases (apatite or OCP) relative to disordered phases (ACP). The first phase transition was complete within 15 min followed by a much later transition. In order to better elucidate the nature of these phase transitions, the OD data was plotted alongside the ATR-FTIR data in Figure 6. Interestingly, the sNP to nanorod transition


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observed in the OD data occurred between the two phase transitions observed by ATR-FTIR. Therefore, in the observed mineralization process, starting from dissolved ions and resulting in suspended apatite nanorods, we find four distinct transitions, summarized in Table 1. These transitions are based upon processes corresponding to change in particle phase or aggregation state.

Figure 6: The ratio of the 1032 cm-1 and 1076 cm1

peaks are compared to the optical density of the

samples from the spectrophotometer experiments for the 30 mg/mL pAsp 50 mM Ca2+ sample over 12 hours. The arrows indicate the transition regions (T2, T3, T4) observed using the two techniques. The results show that the changes in the aggregate size/shape, as determined from the OD data (T3), is completed before the changes in


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the phosphate, as determined from the ATR data (T4). Table 1: Four measured transitions in the mineralization process Transition 1

Process Polymer stabilized spherical nanoparticles

Evidence OD and cryo-TEM data indicated that

(sNP) formed rapidly after mixing (

Polyaspartic Acid Concentration Controls the Rate of Calcium

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