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Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system
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Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system Fioleda P. Kesseli , Caroline S. Lauer , Ian Baker , Katherine A. Mirica , Douglas W. Van Citters PII: DOI: Reference:
S1742-7061(20)30008-8 https://doi.org/10.1016/j.actbio.2020.01.007 ACTBIO 6530
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Acta Biomaterialia
Received date: Revised date: Accepted date:
25 October 2019 14 December 2019 8 January 2020
Please cite this article as: Fioleda P. Kesseli , Caroline S. Lauer , Ian Baker , Katherine A. Mirica , Douglas W. Van Citters , Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system, Acta Biomaterialia (2020), doi: https://doi.org/10.1016/j.actbio.2020.01.007
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Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system Fioleda P. Kesseli, Caroline S. Lauer, Ian Baker, Katherine A. Mirica, Douglas W. Van Citters Thayer School of Engineering, Dartmouth College, Hanover, NH
Abstract Calcium phosphate-based bone cements have been widely adopted in both orthopedic and dental applications. Phosphoserine (pSer), which has a natural role in biomineralization, has been identified to possess the functionality to react with calcium phosphate phases, such as tetracalcium phosphate (TTCP) and α-tricalcium phosphate (α-TCP), and form a uniquely adhesive cement. This study investigated the chemical composition and phase evolution of a heterogeneous calcium phosphate (56% TTCP and 15% α-TCP) and pSer cement system with respect to pH. The coordination network of calcium phosphoserine monohydrate was discovered as the predominant crystalline phase of this adhesive apatitic cement system. Furthermore, it was determined that pH has a significant effect on the reaction kinetics of the system, whereby a lower pH tends to accelerate the reaction rate and favor products with lower Ca/P ratios. These findings provide a better understanding of the reaction and products of this adhesive organo-ceramic cement, which can be compositionally tuned for broad applications in the orthopedic and dental spaces.
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Statement of Significance CPS The application of self-setting calcium phosphate cements (CPCs) in hard tissue regeneration has been a topic of significant research since their introduction to the field 30 years ago. Traditional CPCs, however, are limited by their suboptimal mechanical properties due to their solely inorganic composition. Recently, it was discovered that monomeric phosphoserine (pSer) is capable of serving as a setting reagent for a subset of CPC systems, resulting in an adhesive organo-ceramic composite. Despite its adhesive functionality and biomedical potential, its reaction chemistry and product composition were not well characterized. The present study identifies a calcium phosphoserine coordination network as the primary crystalline phase of this apatitic cement system and further characterizes compositional tunability of the products with respect to pH.
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1. Introduction Calcium phosphate (CaP) biomaterials are readily used in the medical field due to their compositional similarity to bone mineral, biocompatibility, resorbability, and osteoconductivity. Their medical indications range from osteoconductive coatings on implant surfaces to stand-alone bone fillers and synthetic grafting materials for bone loss. Self-setting CaP cements, in particular, have been widely adopted due to their ease of use and tailorability. These cements are primarily synthesized through the reaction of one or more inorganic phases in the presence of water; the dissolution and subsequent precipitation reactions result in mineral crystallization of the products and final cement hardening.[1-3] The suboptimal mechanical properties of these traditional CaP cements, however, have limited their expansion to load-bearing applications (e.g. fracture fixation, dental implantation, etc.). Unlike bone, calcium phosphate cements have poor tensile and shear properties due to the brittle nature of their inorganic chemistry. [4-7] There remains a need for biomaterials which incorporate both inorganic and organic components that can be tailored to mimic the composition and physical properties of bone. It was recently discovered that the amino acid phosphoserine (pSer) is also capable of reacting with certain calcium phosphate phases (e.g. TTCP—tetracalcium phosphate and α-TCP—α-tricalcium phosphate) to form a unique biocompatible organo-ceramic self-curing cement with adhesive properties.[8, 9] Phosphoserine had been previously identified as a chemical motif in several biological marine adhesives, which attracted enthusiasm about its molecular properties.[8, 10-13] Additionally, the discovery of pSer in several biological fluids (i.e. blood, urine, milk, and saliva) has fueled interest regarding its role in biomineralization. [14-17] Despite these reports of its biological significance and adhesive functionality, its chemical reaction with calcium phosphate and resulting product composition are still not well understood.[9, 18-24]
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It is hypothesized that the dual chemical reactivity of pSer (Brønsted-Lowry acid and Lewis base) in the presence of CaP phases, can result in a multiphasic cement system containing traditional inorganic CaP products as well as organo-ceramic coordination networks. In the present work, Tetracalcium phosphate (TTCP; Ca4(PO4)2O) was chosen as the primary crystalline CaP reactant phase due to its optimal chemical properties, including its alkalinity (allowing for acid-base reactions) and high calcium to phosphate (Ca/P) ratio of 2.0, relative to biologically-derived hydroxyapatite (HA; Ca5(PO4)3OH) with a Ca/P ratio of 1.67. [1, 25-27] Furthermore, the reaction between pSer and TTCP has been reported to occur within a medically relevant timescale, initiating within seconds and curing after several minutes.[8] The objective of the present work is to characterize the chemical reaction between an optimized TTCP mixture (containing 56% TTCP, 15% α-TCP, and 29% amorphous/nano-crystalline calcium phosphate—ACP) and pSer in a dilute and controlled environment in order to understand the composition of reaction products and their evolution with respect to pH over a longer timescale. 2. Materials and Methods 2.1 Materials TTCP mixture was obtained custom-made directly from the manufacturer (Launchpad Medical; Lowell, MA). Briefly, the material was synthesized by a solid-state reaction containing a slurry of calcium carbonate and DCPA (9:10 molar ratio), yielding a product with a phase purity of 56.2 wt. % TTCP, 15.2% α-TCP, and 28.6% ACP. Note that this composition of TTCP mixture was chosen based on the company’s reported optimized adhesive properties and reaction rate relevant to its medical applications. The product was mechanically milled and sieved to an average particle size of 12.0 μm ± 7.2 μm. X-Ray diffraction (XRD) patterns for the TTCP mixture, as well as pure TTCP (COD# 9011144), α-TCP (COD# 2106194), and HA (COD# 9002215) are shown for reference in Figure 1. Phosphoserine (Flamma S.p.A.) was obtained as a dry, white powder.
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Figure 1: TTCP mixture compared to reference patterns for pure TTCP, α-TCP, and HA.
2.2 Titration Experimental Design Potentiometric titration was chosen as a method to mimic the cementitious acid-base reaction of this system in a relatively dilute environment.[28] The effects of pH on the reaction kinetics of the TTCP mixture and pSer were determined via a pH-stat technique (Figure 2). The timescale of the titration experiment was Figure 2: Schematic of pH-stat titration.
designed to capture the reaction profile and
phase evolution intermediates and final products. The reactions were conducted at pH end points of 6.0, 7.0, and 8.0, at 37 ᵒC. These values were chosen to capture the reaction kinetics at and around physiologic pH 7.4 and temperature. Each pH value was tested in triplicate. The experimental design
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was modified from Chow et al. 2005.[28] A pH electrode was calibrated in standard pH solutions (4.0, 7.0, and 10.0) prior to each titration. The titration reaction solution used 100 mL of 0.15 M KNO3, and was pH-equilibrated (i.e., CO2 displaced) with laboratory grade N2 gas bubbled into the covered solution at a rate of 34 L/min prior to each reaction. TTCP mixture (1.0 g) was added to the reaction solution and the titration was started immediately. The titrant containing 0.15 M pSer in 0.15M KNO3, was dispensed by an automatic titrator (TitroLine 7000—Xylem, Inc.) with a dosing step size of 10 μl, until the objective pH was achieved. pH was maintained constant for the remainder of the reaction time with additional titrant dosing as needed. The reaction was considered complete when no additional pSer was necessary to maintain the objective pH for at least 1h. At the completion of each titration, the product was divided; one part was used for direct analysis (described later) and the other part was kept in its reaction solution for 7 days of incubation at 37 ᵒC. All titrations were conducted in triplicate. 2.3 Product Evolution A second series of titrations (n=19) were conducted to monitor reaction progress and product evolution at pH 6.0, 7.0, and 8.0. Reactions were terminated at no fewer than six varying time points for each pH as shown in Table 1. Time points were selected based on the shape of the titration curves established in the aforementioned experiment.
Table 1: Time points for phase evolution study. Rounded to nearest hour, except if under 1h. pH Time points (h) 6
0.8, 2, 3, 4, 5, 6, 15
7
0.7, 4, 9, 13, 17, 29
8
4, 9, 21, 39, 51, 80
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2.4 Calcium Phosphoserine Phase Synthesis A pH-stat titration of calcium hydroxide, 1.0 mM Ca(OH)2 with 0.15 M pSer titrant was conducted at pH 8, under N2 atmosphere at 37ᵒC. The goal of this titration was to synthesize the pure coordination compound of calcium phosphoserine (CPS) according to the following chemical reaction. Ca(OH)2 + C3H8NO6P [Ca(C3H6NO6P)(H2O)] + H2O
2.5 Product Characterization The precipitated product from each titration and/or incubation was filtered, rinsed with DI water, and air dried prior to characterization by X-ray diffraction (XRD), FT-IR, and SEM. XRD (Rigaku 007, 40kV/300mA, 2Ө: 10-60ᵒ, step size: 0.02ᵒ, dwell time: 5 s) was performed on each product with an internal standard of corundum (approximately 10 wt.%). Each diffraction pattern was tested for the presence of calcium phosphate phases as well as any other matched phases identified from the Crystallography Open Database (COD-2018). Jade (MDI) Whole Pattern Fitting (WPF) analysis was applied on matched phases using exact corundum values, and the quantified results of each set of experimental trials were averaged to determine mass percent (%) composition. ACP content was determined by the software based on percentage of unmatched and non-crystalline phases. FT-IR spectroscopy (Perkin Elmer Spectrum 100) was used as a secondary qualitative validation of phase composition to corroborate quantitative XRD results. Powder samples were prepared using FT-IR grade potassium bromide (KBr). Approximately 1 % (w/w) of sample was mixed with 99% KBr with an agate mortar and pestle and consolidated into pellets using a stainless steel 7 mm die kit, and a table-
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top hand press (International Crystal Laboratories). Each test included 64 scans (4000-400 cm-1). Characteristic functional groups were identified based on reported absorbance ranges ( Table 2) for similar organic and inorganic compounds. [29-31] Table 2: Relevant FT-IR characteristic group frequencies Functional Group OH st H2O Amines N-H st NH2 δ Carboxylic Acids COO-H st C=O st Phosphates v3PO4 v1PO4 v4PO4 v2PO4
Absorption Range (cm-1) 3200-3650 3000-3700 3300-3500 1550-1650 2500-3550 1650-1800 1000-1150 ~950 500-620 400-470
Scanning electron microscopy (SEM) was also used as an aid for phase identification for the phase evolution study. Dry samples were loaded onto carbon tape and sputter coated (Anatech, Hummer V Sputter Coater) with gold, for 2 min at 15 mA and ~90 mTorr, resulting in a coating thickness of about 10 nm. Imaging was performed using a FEI Scios2 SEM, with the following conditions: HV 5.0 kV, WD ≤ 7 mm, and an ET (mixed secondary and backscatter) detector. Crystal structure and morphology, as well as phase interactions were visualized. 3. Results 3.1 Titration Parameters Figure 3 shows the titration reaction curves, monitoring pSer consumption as a function of time, at pH values of 6.0, 7.0, and 8.0. The consumption was normalized to 1 mol of pure TTCP. The three reaction
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trials for each pH value were all consistent and reproducible, and each show a characteristic double logistic curve.
Figure 3: Titration curves for pH comparison of pSer and TTCP reaction. Y-axis normalized to 1 mol TTCP.
Titration parameters were calculated and are summarized in Error! Reference source not found.. At the beginning of each titration, the initial dissolution of some TTCP caused an immediate increase in pH, and consequently, a bulk addition of titrant was observed until the preset pH was reached. This initial consumption of titrant was omitted from the rate calculations but included in the total consumption. The consumption and time to reach the desired pH are noted as Ci and ti, respectively. For titration curves with observed inflections, the rate of titration for each region of the graph was calculated individually. The maximum rate for the first part of the reaction (R1) was calculated from the first consistent slope after ti. The second maximum rate of consumption (R2), was calculated from the second increase in consumption after the middle plateau region. The minimum (Rmin) rate of titration for the plateau region was also calculated. The corresponding times for each of these rate calculations are 9
noted below the rate. Completion time for the reaction (tf) was determined as the initial time at which the titrant consumption reached its maximum (Cf) and remained stable for at least 1 h. Table 3: Reaction parameters for pH comparison titrations.
pH 6
Parameters C (mol) R (mol/h) Time (h)
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C (mol) R (mol/h) Time (h)
8
Ci 0.54 0.02 0.19 0.02 0.08 0.04 0.06 0.04
C (mol) R (mol/h)
-
Time (h)
-
R1 0.28 0.02 0.29 0.02 0.10 0.01 0.51 0.02 0.04 0.00 1.00 0.26
R2 0.18 0.01 5.26 0.73 0.04 0.01 12.07 1.37 0.01 0.00 32.73 8.71
Rmin 0.03 0.01 0.92 0.08 0.02 0.00 7.71 0.60 0.004 0.00 15.87 2.29
Cf 1.29 0.02 11.79 1.12 0.85 0.01 33.52 2.02 0.68 0.03 58.40 1.28
From Error! Reference source not found., it can be seen that Cf, R1, and R2 are inversely related to pH. The product of pH 6 consumed nearly twice as much pSer as that of pH 8, and the reaction was complete in 80% less time. The maximum reaction rates, R1 and R2 (corresponding to the initial and second growth curves), decrease with pH, and in all cases R1 is greater than R2. The peak rate of the second growth curve (R2) is reached after 72% total consumption for pH 6, 68% for pH 7, and 60% for pH 8.
3.2 Product Characterization Figure 3 summarizes the average XRD patterns (from three trials) for each pH at t=0 d and t=7 d. The patterns confirmed the presence of two primary crystalline phase products, hydroxyapatite (HA)
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and calcium phosphoserine monohydrate complex (CPS, Figure 4), in addition to the amorphous content. The measured α-TCP, which represented only 15 wt% of the starting material, was completely consumed at the one week time point for all pH values, as can be seen in the diffractograms (Figure 5, dotted drop line). Considering that the reactions of both TTCP and α-TCP with pSer yielded the same two phases after 7 days, the effects of residual α-TCP at t=0 were deemed negligible for phase analysis.
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Figure 5: Average XRD patterns for titrations conducted under pH values of 6, 7, and 8, with incubation time points of 0 and 7 days. CPS marked by solid circle, HA by solid triangle, α-TCP by open circle (and dashed drop line), and corundum internal standard by asterisk. Y-axis is arbitrary units (AU) of intensity; plots are y-offset for clarity.
Figure 4: XRD pattern of titration product of CPS reaction. CPS marked by green lines, Corundum standard peaks marked by blue lines and asterisk.
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The CPS synthesized by the Ca(OH)2 reaction in the present work was determined to be highly crystalline and phase pure, as shown in the diffractogram in Figure 4, with colored lines noting matched phases from the COD database. Qualitative FT-IR analysis (Figure 6) of chemically pure CPS as well as the titration products supports the presence of both HA and CPS, specifically with the phosphate vibrations in the fingerprint region and distinct peaks highlighting the presence of both primary amines and carboxyl groups. The relative intensities of the carboxyl and amine peaks decrease with pH, while the phosphate peaks broaden to closely match the spectrum of HA. Figure 7 shows a representative SEM image of each titration product, including the pure CPS product. Figure 8 summarizes the phase composition (mol %) assuming stoichiometric formulas of the titration products showing CPS as the primary phase at every pH value and time point. Percent crystalline product is the sum of CPS and HA, compared to the amorphous and/or nanocrystalline content represented by ACP.
Figure 6: FT-IR spectra of titration products from pSer and TTCP at pH 6, 7, and 8. CPS and HA shown for reference. Relevant functional group ranges shown per Table 2.
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Figure 7: SEM images of (A) CPS, (B) pH 6, (C) pH 7, and (D) pH 8 titration products.
Figure 8: Phase composition (mol%) of titration reaction products for pH 6, 7, and 8 at time points t=0d and t=7d.
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3.4 Product Evolution The phase evolution of the titration reaction products at pH 6, 7 and 8 were also analyzed by XRD and whole pattern fitting, the results of which (moles solid products) are summarized in Figure 9, overlaid with each titration curve. At the completion of the initial growth curve, the main product formed is ACP, with a small amount of HA. At every pH, detection of CPS corresponds with the second growth curve with an initially high rate of formation at that time point. In contrast, the crystallization and precipitation of HA is much more gradual. The time series of SEM images graphically depicts this phase evolution as a function of time. Figure 10 shows an initial formation of ACP (beaded structure), followed by the precipitation and growth of the monoclinic blooming crystals of CPS, as well as the foam-like characteristic growth of HA. Both ACP and HA have been previously confirmed to have these structural features in the presence of organic molecules. [23, 32-34]
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Figure 9: Phase evolution (by moles) of titration product composition at pH 6, 7 and 8. Product composition based on total solid retrieved at each time point less any unreacted components. Theoretical stoichiometry was assumed for molar conversion.
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Figure 10: SEM images of phase evolution of pSer titration at pH 6. Phases are labeled as ACP (A), CPS (C) and HA (H). 4. Discussion 4.1 Titration Reactions It is well established that HA is the most common CaP cement product due to its thermodynamic stability in the pH range of 6-8. TTCP has been commonly used in numerous traditional calcium phosphate cements. Most of these cements are composed of purely inorganic phases, usually forming HA or brushite[3]. The results of the titration experiments in this study are consistent with those of a similar study by Chow et al. which examined the effects of pH on reaction kinetics and product 17
composition of a reaction between TTCP and phosphoric acid.[28] In the pH range relevant to this study (pH 7 and 8), they found that the reaction kinetics were mostly limited by TTCP dissolution, because the solution was supersaturated with HA and undersaturated with TTCP. Chow et al. also reported affects of particle size on reaction rate, concluding that smaller TTCP particles accelerated the overall rate. In the present study, the use of a TTCP mixture containing α-TCP and ACP, reduced the relative rate of the reaction. The use of pure TTCP would have increased the pH of the overall reaction due to it high solubility and alkalinity and would have required greater volumes of pSer at an accelerated dispensing rate to maintain the set pH. The heterogeneous mixture of TTCP and α-TCP allowed for an extended reaction time due to α-TCP’s slower dissolution at the given pH range.[35] Unlike the Chow et al. study with phosphoric acid, which produced a single logarithmic growth curve, the present study resulted in a characteristic growth curve with two inflections, suggesting that pSer plays a dual role and results in two distinct reaction pathways. In contrast to phosphoric acid, pSer has the reactivity of both a Bronsted-Lowry acid as well as a Lewis base. This dual reactivity is believed to contribute to the double logisitic curve as well as the formation of CPS, the predominant phase in this adhesive cement system. CPS was first reported by Suga and Okabe in 1996, when they synthesized the compound using an evaporation method from a 5:1 molar mixture of pSer and CaCl2 ∙ 2H2O at pH 7.[36] Their characterization of CPS, which they referred to by its coordinate compound name, aqua (L-O-serine phosphato) calcium (II), was limited to the identification of its crystal structure. Interestingly, in CPS, pSer does not act as a chelating ligand (i.e. one ligand donating multiple electron pairs to a single metal ion) as was previously theorized in the literature about its Ca2+-binding mechanism.[24, 37-41] Instead, each pSer molecule bonds to five distinct calcium ions (each in a monodentate manner), serving as a bridging ligand.[42] Although not previously identified as such, CPS meets the criteria for being a coordination network, as modeled in Figure 11. 18
Despite its chemical identification 20 years ago, the potential link between CPS and biological mineralization has not yet been explored. A study by Wu et. al. confirmed the presence of general calcium-organic phosphate complexes in early stages of bone mineralization, suggesting a potential role for molecules like CPS in the initiation of bone calcification. [43] A recent study by Kirillova et al. reported the mechanical and biological properties of the TTCP-pSer cement system, however, the chemical composition of the cement was not characterized.
They found that in addition to its
osteoconductive properties the cement product also exhibited adhesive properties, suggesting the presence of CPS. [8] Another recent publication sought to determine the molecular basis of adhesion for the reaction products of pSer and α-TCP [9]. Despite the presence of CPS in their XRD data, it was not identified nor discussed, rather several of its peaks were erroneously attributed to α-TCP. Based on the results of the present study and the identification of CPS as the main component of the TTCP-pSer system, it is likely that the self-assembly of the chemically continuous structure of its coordination network gives rise to the unique physical properties and adhesive/cohesive interactions reported by both Kirillova et al. and Pujari-Palmer et al.
Figure 11: Three dimensional model of CPS crystal structure. (CrystalMaker Software)
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4.2 Product Evolution The product evolution experiments give further insight into the progress of the reaction and formation of the individual phases in this cement system. This study (Figures 9 and 10) suggests that the initial growth curve is representative of TTCP dissolution, which leads to a rapid increase in alkalinity. In the absence of pSer, and the use of phosphoric acid as a titrant, Chow et al. documented a single growth curve with TTCP dissolution coinciding with the precipitation of the HA product. However, in this system, it is proposed that the initial asymptote is the result of pSer adsorption onto both TTCP and ACP, which slows dissolution of TTCP. Similar stabilizing effects have been previously reported for calcium phosphates and calcium carbonates in the presence of pSer and other organic molecules.[18-20] For instance, in Figure 10, at the 0.8 h timepoint at pH 6, the TTCP particles are already covered in amorphous/nano-crystalline phases, limiting diffusion. This decreased dissolution as well as the possible inhibition of HA crystallization creates a temporal shift in the classic HA-forming reaction. This hypothesis is supported by the abundance of ACP, which is a known precursor phase for HA; its presence in the product indicates that the formed HA is partially crystalline—broad peaks caused by small, nano-scale crystals. This observation has been also made by others in the literature.[28, 44] The presence of organic entities is also known to produce less crystalline and non-stoichiometric HA.[28, 45, 46] Despite the heterogeneous composition of the starting TTCP mixture, containing α-TCP and ACP, it is expected that in the pH range of 6-8 and at 37C, the two thermodynamically stable phases for the pSer and CaP system are CPS and HA. CaP chemistry has well established the phase diagrams for the Ca-PH2O at various temperatures and pH values.[35] Similarly, an understanding of pSer speciation with respect to pH supports the formation of CPS due to the high abundance of pSerH2- in the pH range of 68.[47]
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Based on evidence from the titration curves and materials characterization, we propose the following reaction pathways for the TTCP-pSer organo-ceramic system in the physiologic pH range:
3Ca4(PO4)2O + 3H2O 3Ca10(PO4)6(OH)2 + 2CA(OH)2 HA Formation
a4(PO4)2O + 3H2O + 4C3H8NO6P 4[Ca(C3H6NO6P)(H2O)] + 2(H3PO4) CPS Formation
These pathways, although shown in parallel, are interdependent due to their common reactant (TTCP) as well as their potentially reactive by-products. For example, Ca(OH)2, produced during HA formation, can also react with pSer to from CPS. Similarly, phosphoric acid, produced during CPS formation, can also react with TTCP to form HA and other CaP phases. The pH of the system can further favor or hinder each pathway based on the solubility of both intermediate and final products. As a biomaterial, this cement system is designed to be mixed and injected for curing in situ. In a physiologically buffered environment, the system becomes increasingly complex due to the potential participation of other ions in the reaction. The fast curing time (as reported by Kirillova et al), may initially hinder the diffusion of many of these ions within the cement, however, over time it is expected that the cement will evolve with the surrounding mineral environment in a similar fashion as biological HA. The local pH of the cement system as it cures in vivo is also expected to vary based on the pSer:TTCP ratio, which might in turn affect the phase composition of the final product. Although HA and CPS are the expected products at physiological pH, a lower pH, for example, might result in brushite or a different coordination network of pSer and calcium ions. The present study provides insight into the 21
unique CPS coordination network which forms under a subset of controlled conditions mimicking this cement system and suggests a more complex family of organo-ceramic phases which likely form under varying pH and ionic conditions. 5. Conclusions The primary purpose of this work was to better understand the reaction kinetics and chemical composition of a novel class of cements which are currently being clinically developed. In contrast to the inorganic components of traditional CaP cement and their potential organic additives, the present study revealed that both TTCP and pSer chemically participate in a set of interdependent reaction pathways that lead to a uniquely adhesive composite cement system. The effects of pSer on the reaction kinetics of HA crystallization is believed to result in the temporal shift observed in the signature double logistic curve of the reaction profile for this system. It was determined that pH has a significant effect on the reaction rate of TTCP-pSer, however, the system’s products are robust within the pH range of 6 and 8. Phosphoserine’s participation in this reaction is a result of its dual functionality as both a Brønsted-Lowry acid and Lewis base. Furthermore, this work revealed the previously unidentified presence of the coordination network, CPS, in an apatitic cement system. This crystalline compound was found to be the predominant phase for this system at all tested pH values. The fundamental findings presented here, better prepare our group for future functional mechanical testing and biological assessment of this system. The unique multiphasic composition of this system may provide insight into the complexities of bone mineralization as well as set the stage for further development CPS-based biomaterials and cements with functional physical properties.
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F.P.K. and C.S.L performed the titrations. F.P.K., C.S.L., and D.W.VC. designed the experiments. F.P.K. wrote the paper. All authors analyzed data and edited paper. We thank Dr. Laurence C. Chow for useful discussions about calcium phosphate chemistry. This research has been supported by a Small Business Technology Transfer grant from the National Institute on Aging of the U.S. National Institutes of Health, Award Number R41AG060881. Declaration of interests None
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References [1] L.C. Chow, Development of Self-Setting Calcium-Phosphate Cements, Nippon Seram Kyo Gak 99(10) (1991) 954-964. [2] L.C. Chow, E.D. Eanes, Octacalcium phosphate, Karger, Basel ; New York, 2001. [3] M. Bohner, Reactivity of calcium phosphate cements, Journal of Materials Chemistry 17(38) (2007) 3980. [4] K.L. Low, S.H. Tan, S.H. Zein, J.A. Roether, V. Mourino, A.R. Boccaccini, Calcium phosphate-based composites as injectable bone substitute materials, J Biomed Mater Res B Appl Biomater 94(1) (2010) 273-86. [5] H.H. Xu, J.B. Quinn, Calcium phosphate cement containing resorbable fibers for short-term reinforcement and macroporosity, Biomaterials 23(1) (2002) 193-202. [6] J.M. Cervantes-Uc, J.V. Cauich-Rodríguez, F. Hernández- Sánchez, L.H. Chan-Chan, Bone Cements: Formulation, Modification, and Characterization, Encyclopedia of Biomedical Polymers and Polymeric Biomaterials, Taylor and Francis, New York, 2015, pp. 1053-1066. [7] M. Bohner, Design of ceramic-based cements and putties for bone graft substitution, Eur Cell Mater 20 (2010) 1-12. [8] A. Kirillova, C. Kelly, N. von Windheim, K. Gall, Bioinspired Mineral-Organic Bioresorbable Bone Adhesive, Adv Healthc Mater 7(17) (2018) e1800467. [9] M. Pujari-Palmer, H. Guo, D. Wenner, H. Autefage, C.D. Spicer, M.M. Stevens, O. Omar, P. Thomsen, M. Eden, G. Insley, P. Procter, H. Engqvist, A Novel Class of Injectable Bioceramics that Glue Tissues and Biomaterials, Materials (Basel) 11(12) (2018). [10] J.B. Addison, W.S. Weber, Q. Mou, N.N. Ashton, R.J. Stewart, G.P. Holland, J.L. Yarger, Reversible assembly of beta-sheet nanocrystals within caddisfly silk, Biomacromolecules 15(4) (2014) 1269-75. [11] V. Bhagat, E. O'Brien, J. Zhou, M.L. Becker, Caddisfly Inspired Phosphorylated Poly(ester urea)-Based Degradable Bone Adhesives, Biomacromolecules 17(9) (2016) 3016-24. [12] R.J. Stewart, J.C. Weaver, D.E. Morse, J.H. Waite, The tube cement of Phragmatopoma californica: a solid foam, J Exp Biol 207(Pt 26) (2004) 4727-34. [13] H. Zhao, C. Sun, R.J. Stewart, J.H. Waite, Cement proteins of the tube-building polychaete Phragmatopoma californica, J Biol Chem 280(52) (2005) 42938-44. [14] A. Bennick, A.C. McLaughlin, A.A. Grey, G. Madapallimattam, The location and nature of calciumbinding sites in salivary acidic proline-rich phosphoproteins, J Biol Chem 256(10) (1981) 4741-6. [15] D.I. Hay, E.R. Carlson, S.K. Schluckebier, E.C. Moreno, D.H. Schlesinger, Inhibition of calcium phosphate precipitation by human salivary acidic proline-rich proteins: structure-activity relationships, Calcif Tissue Int 40(3) (1987) 126-32. [16] M. Johnsson, C.F. Richardson, E.J. Bergey, M.J. Levine, G.H. Nancollas, The effects of human salivary cystatins and statherin on hydroxyapatite crystallization, Arch Oral Biol 36(9) (1991) 631-6. [17] M. Arifin, P.J. Swedlund, Y. Hemar, I.R. McKinnon, Calcium phosphates in Ca(2+)-fortified milk: phase identification and quantification by Raman spectroscopy, J Agric Food Chem 62(50) (2014) 122238. [18] T. Aoba, E.C. Moreno, Adsorption of phosphoserine onto hydroxyapatite and its inhibitory activity on crystal growth, Journal of colloid and interface science 106(1) (1985) 110-121. [19] L. Benaziz, A. Barroug, A. Legrouri, C. Rey, A. Lebugle, Adsorption of O-Phospho-L-Serine and LSerine onto Poorly Crystalline Apatite, J Colloid Interface Sci 238(1) (2001) 48-53. [20] S. Bentov, S. Weil, L. Glazer, A. Sagi, A. Berman, Stabilization of amorphous calcium carbonate by phosphate rich organic matrix proteins and by single phosphoamino acids, J Struct Biol 171(2) (2010) 207-15. 24
[21] L. Offer, B. Veigel, T. Pavlidis, C. Heiss, M. Gelinsky, A. Reinstorf, S. Wenisch, K.S. Lips, R. Schnettler, Phosphoserine-modified calcium phosphate cements: bioresorption and substitution, J Tissue Eng Regen Med 5(1) (2011) 11-9. [22] A. Reinstorf, M. Ruhnow, M. Gelinsky, W. Pompe, U. Hempel, K.W. Wenzel, P. Simon, Phosphoserine--a convenient compound for modification of calcium phosphate bone cement collagen composites, J Mater Sci Mater Med 15(4) (2004) 451-5. [23] M.T. Jahromi, G. Yao, M. Cerruti, The importance of amino acid interactions in the crystallization of hydroxyapatite, J R Soc Interface 10(80) (2013) 20120906. [24] M. Tavafoghi, M. Cerruti, The role of amino acids in hydroxyapatite mineralization, J R Soc Interface 13(123) (2016). [25] E.F. Burguera, F. Guitián, L.C. Chow, A water setting tetracalcium phosphate-dicalcium phosphate dihydrate cement, Journal of Biomedical Materials Research Part A 71A(2) (2004) 275-282. [26] Y. Fukase, E.D. Eanes, S. Takagi, L.C. Chow, W.E. Brown, Setting reactions and compressive strengths of calcium phosphate cements, J Dent Res 69(12) (1990) 1852-6. [27] C. Hamanishi, K. Kitamoto, K. Ohura, S. Tanaka, Y. Doi, Self-setting, bioactive, and biodegradable TTCP-DCPD apatite cement, J Biomed Mater Res 32(3) (1996) 383-9. [28] L.C. Chow, M. Markovic, S.A. Frukhtbeyn, S. Takagi, Hydrolysis of tetracalcium phosphate under a near-constant-composition condition--effects of pH and particle size, Biomaterials 26(4) (2005) 393-401. [29] C. Rey, C. Combes, C. Drouet, D. Grossin, Bioactive Ceramics: Physical Chemistry, Comprehensive Biomaterials, Elsevier2011. [30] C. Drouet, Apatite formation: why it may not work as planned, and how to conclusively identify apatite compounds, Biomed Res Int 2013 (2013) 490946. [31] E. Pretsch, P. Buhlmann, M. Badertscher, Structure and Determination of Organic Compounds: Tables of Spectral Data, 4 ed., Springer, Berlin, 2009. [32] A.C. Tas, X-ray-amorphous calcium phosphate (ACP) synthesis in a simple biomineralization medium, J Mater Chem B 1(35) (2013). [33] A.C. Tas, Calcium metal to synthesize amorphous or cryptocrystalline calcium phosphates, Materials Science and Engineering: C 32(5) (2012) 1097-1106. [34] T. Welzel, W. Meyer-Zaika, M. Epple, Continuous preparation of functionalised calcium phosphate nanoparticles with adjustable crystallinity, Chem Commun (Camb) (10) (2004) 1204-5. [35] L.C. Chow, Next generation calcium phosphate-based biomaterials, Dent Mater J 28(1) (2009) 1-10. [36] T. Suga, N. Okabe, Aqua(L-O-serine phosphato)calcium(II), Acta Crystallogr C 52 (1996) 1894-1896. [37] M.S. Mohan, E.H. Abbott, Metal-Complexes of Amino-Acid Phosphate Esters, Inorganic Chemistry 17(8) (1978) 2203-2207. [38] M.S. Mohan, D. Bancroft, E.H. Abbott, Stereoselectivity in the Formation of the Metal Complexes of O-Phosphoserine, Inorg Chem 22(5) (1982) 714-721. [39] M.S. Mohan, D. Bancroft, E.H. Abbott, Mixed-Ligand Complexes of O-Phospho-Dl-Serine, Inorganic Chemistry 18(9) (1979) 2468-2472. [40] H.S. Hendrickson, J.G. Fullington, Stabilities of Metal Complexes of Phospholipids: Ca(II), Mg(II), and Ni(I1) Complexes of Phosphatidylserine and Triphosphoinositide, Biochemistry 4(8) (1965) 1599-1605. [41] M. Zachariou, I. Traverso, L. Spiccia, M.T.W. Hearn, Potentiometric investigations into the acid-base and metal ion binding properties of immobilized metal ion affinity chromatographic (IMAC) adsorbents, Journal of Physical Chemistry 100(30) (1996) 12680-12690. [42] Coordination Compounds, in: N.G. Connelly (Ed.) Nomenclature of Inorganic Chemistry - Revised 'Red Book', International Unit of Pure and Applied Chemistry, 2004. [43] Y. Wu, J. Ackerman, E. Strawich, C. Rey, H.-M. Kim, M. Glimcher, Phosphate ions in bone: identification of a calcium-organic phosphate complex by 31P solid-state NMR spectroscopy at early stages of mineralization, Calcified Tissue Int 72(5) (2003) 610-626. 25
[44] K. Onuma, A. Ito, Cluster growth model for hydroxyapatite, Chem Mater 10(11) (1998) 3346-3351. [45] M. Öner, Ö. Doğan, Inhibitory effect of polyelectrolytes on crystallization kinetics of hydroxyapatite, Prog Cryst Growth Ch 50(1-3) (2005) 39-51. [46] S. Koutsopoulos, Synthesis and characterization of hydroxyapatite crystals: a review study on the analytical methods, J Biomed Mater Res 62(4) (2002) 600-12. [47] Q.-Y. Wu, C.-Z. Wang, J.-H. Lan, Z.-F. Chai, W.-Q. Shi, Theoretical insight into the binding affinity enhancement of serine with the uranyl ion through phosphorylation, RSC Adv. 6(74) (2016) 6977369781.
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Figure Captions List
Figure 1: TTCP mixture compared to reference patterns for pure TTCP, α-TCP, and HA. Figure 2: Schematic of pH-stat titration. Figure 3: Titration curves for pH comparison of pSer and TTCP reaction. Y-axis normalized to 1 mol TTCP. Figure 4: Average XRD patterns for titrations conducted under pH values of 6, 7, and 8, with incubation time points of 0 and 7 days. CPS marked by solid circle, HA by solid triangle, α-TCP by open circle (and dashed drop line), and corundum internal standard by asterisk. Y-axis is arbitrary units (AU) of intensity; plots are y-offset for clarity. Figure 5: XRD pattern of titration product of CPS reaction. CPS marked by green lines, Corundum standard peaks marked by blue lines and asterisk. Figure 6: FT-IR spectra of titration products from pSer and TTCP at pH 6, 7, and 8. Figure 7: SEM images of (A) CPS, (B) pH 6, (C) pH 7, and (D) pH 8 titration products. Figure 8: Phase composition (mol%) of titration reaction products for Figure 9: Phase evolution (by moles) of titration product composition at pH 6, 7 and 8. Product composition based on total solid retrieved at each time point less any unreacted components. Theoretical stoichiometry was assumed for molar conversion. Figure 10: SEM images of phase evolution of pSer titration at pH 6. Phases are labeled as ACP (A), CPS (C) and HA (H). Figure 11: Three dimensional model of CPS crystal structure. (CrystalMaker Software)
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Graphical Abstract
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Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system Fioleda P. Kesseli , Caroline S. Lauer , Ian Baker , Katherine A. Mirica , Douglas W. Van Citters PII: DOI: Reference:
S1742-7061(20)30008-8 https://doi.org/10.1016/j.actbio.2020.01.007 ACTBIO 6530
To appear in:
Acta Biomaterialia
Received date: Revised date: Accepted date:
25 October 2019 14 December 2019 8 January 2020
Please cite this article as: Fioleda P. Kesseli , Caroline S. Lauer , Ian Baker , Katherine A. Mirica , Douglas W. Van Citters , Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system, Acta Biomaterialia (2020), doi: https://doi.org/10.1016/j.actbio.2020.01.007
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Identification of a calcium phosphoserine coordination network in an adhesive organo-apatitic bone cement system Fioleda P. Kesseli, Caroline S. Lauer, Ian Baker, Katherine A. Mirica, Douglas W. Van Citters Thayer School of Engineering, Dartmouth College, Hanover, NH
Abstract Calcium phosphate-based bone cements have been widely adopted in both orthopedic and dental applications. Phosphoserine (pSer), which has a natural role in biomineralization, has been identified to possess the functionality to react with calcium phosphate phases, such as tetracalcium phosphate (TTCP) and α-tricalcium phosphate (α-TCP), and form a uniquely adhesive cement. This study investigated the chemical composition and phase evolution of a heterogeneous calcium phosphate (56% TTCP and 15% α-TCP) and pSer cement system with respect to pH. The coordination network of calcium phosphoserine monohydrate was discovered as the predominant crystalline phase of this adhesive apatitic cement system. Furthermore, it was determined that pH has a significant effect on the reaction kinetics of the system, whereby a lower pH tends to accelerate the reaction rate and favor products with lower Ca/P ratios. These findings provide a better understanding of the reaction and products of this adhesive organo-ceramic cement, which can be compositionally tuned for broad applications in the orthopedic and dental spaces.
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Statement of Significance CPS The application of self-setting calcium phosphate cements (CPCs) in hard tissue regeneration has been a topic of significant research since their introduction to the field 30 years ago. Traditional CPCs, however, are limited by their suboptimal mechanical properties due to their solely inorganic composition. Recently, it was discovered that monomeric phosphoserine (pSer) is capable of serving as a setting reagent for a subset of CPC systems, resulting in an adhesive organo-ceramic composite. Despite its adhesive functionality and biomedical potential, its reaction chemistry and product composition were not well characterized. The present study identifies a calcium phosphoserine coordination network as the primary crystalline phase of this apatitic cement system and further characterizes compositional tunability of the products with respect to pH.
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1. Introduction Calcium phosphate (CaP) biomaterials are readily used in the medical field due to their compositional similarity to bone mineral, biocompatibility, resorbability, and osteoconductivity. Their medical indications range from osteoconductive coatings on implant surfaces to stand-alone bone fillers and synthetic grafting materials for bone loss. Self-setting CaP cements, in particular, have been widely adopted due to their ease of use and tailorability. These cements are primarily synthesized through the reaction of one or more inorganic phases in the presence of water; the dissolution and subsequent precipitation reactions result in mineral crystallization of the products and final cement hardening.[1-3] The suboptimal mechanical properties of these traditional CaP cements, however, have limited their expansion to load-bearing applications (e.g. fracture fixation, dental implantation, etc.). Unlike bone, calcium phosphate cements have poor tensile and shear properties due to the brittle nature of their inorganic chemistry. [4-7] There remains a need for biomaterials which incorporate both inorganic and organic components that can be tailored to mimic the composition and physical properties of bone. It was recently discovered that the amino acid phosphoserine (pSer) is also capable of reacting with certain calcium phosphate phases (e.g. TTCP—tetracalcium phosphate and α-TCP—α-tricalcium phosphate) to form a unique biocompatible organo-ceramic self-curing cement with adhesive properties.[8, 9] Phosphoserine had been previously identified as a chemical motif in several biological marine adhesives, which attracted enthusiasm about its molecular properties.[8, 10-13] Additionally, the discovery of pSer in several biological fluids (i.e. blood, urine, milk, and saliva) has fueled interest regarding its role in biomineralization. [14-17] Despite these reports of its biological significance and adhesive functionality, its chemical reaction with calcium phosphate and resulting product composition are still not well understood.[9, 18-24]
3
It is hypothesized that the dual chemical reactivity of pSer (Brønsted-Lowry acid and Lewis base) in the presence of CaP phases, can result in a multiphasic cement system containing traditional inorganic CaP products as well as organo-ceramic coordination networks. In the present work, Tetracalcium phosphate (TTCP; Ca4(PO4)2O) was chosen as the primary crystalline CaP reactant phase due to its optimal chemical properties, including its alkalinity (allowing for acid-base reactions) and high calcium to phosphate (Ca/P) ratio of 2.0, relative to biologically-derived hydroxyapatite (HA; Ca5(PO4)3OH) with a Ca/P ratio of 1.67. [1, 25-27] Furthermore, the reaction between pSer and TTCP has been reported to occur within a medically relevant timescale, initiating within seconds and curing after several minutes.[8] The objective of the present work is to characterize the chemical reaction between an optimized TTCP mixture (containing 56% TTCP, 15% α-TCP, and 29% amorphous/nano-crystalline calcium phosphate—ACP) and pSer in a dilute and controlled environment in order to understand the composition of reaction products and their evolution with respect to pH over a longer timescale. 2. Materials and Methods 2.1 Materials TTCP mixture was obtained custom-made directly from the manufacturer (Launchpad Medical; Lowell, MA). Briefly, the material was synthesized by a solid-state reaction containing a slurry of calcium carbonate and DCPA (9:10 molar ratio), yielding a product with a phase purity of 56.2 wt. % TTCP, 15.2% α-TCP, and 28.6% ACP. Note that this composition of TTCP mixture was chosen based on the company’s reported optimized adhesive properties and reaction rate relevant to its medical applications. The product was mechanically milled and sieved to an average particle size of 12.0 μm ± 7.2 μm. X-Ray diffraction (XRD) patterns for the TTCP mixture, as well as pure TTCP (COD# 9011144), α-TCP (COD# 2106194), and HA (COD# 9002215) are shown for reference in Figure 1. Phosphoserine (Flamma S.p.A.) was obtained as a dry, white powder.
4
Figure 1: TTCP mixture compared to reference patterns for pure TTCP, α-TCP, and HA.
2.2 Titration Experimental Design Potentiometric titration was chosen as a method to mimic the cementitious acid-base reaction of this system in a relatively dilute environment.[28] The effects of pH on the reaction kinetics of the TTCP mixture and pSer were determined via a pH-stat technique (Figure 2). The timescale of the titration experiment was Figure 2: Schematic of pH-stat titration.
designed to capture the reaction profile and
phase evolution intermediates and final products. The reactions were conducted at pH end points of 6.0, 7.0, and 8.0, at 37 ᵒC. These values were chosen to capture the reaction kinetics at and around physiologic pH 7.4 and temperature. Each pH value was tested in triplicate. The experimental design
5
was modified from Chow et al. 2005.[28] A pH electrode was calibrated in standard pH solutions (4.0, 7.0, and 10.0) prior to each titration. The titration reaction solution used 100 mL of 0.15 M KNO3, and was pH-equilibrated (i.e., CO2 displaced) with laboratory grade N2 gas bubbled into the covered solution at a rate of 34 L/min prior to each reaction. TTCP mixture (1.0 g) was added to the reaction solution and the titration was started immediately. The titrant containing 0.15 M pSer in 0.15M KNO3, was dispensed by an automatic titrator (TitroLine 7000—Xylem, Inc.) with a dosing step size of 10 μl, until the objective pH was achieved. pH was maintained constant for the remainder of the reaction time with additional titrant dosing as needed. The reaction was considered complete when no additional pSer was necessary to maintain the objective pH for at least 1h. At the completion of each titration, the product was divided; one part was used for direct analysis (described later) and the other part was kept in its reaction solution for 7 days of incubation at 37 ᵒC. All titrations were conducted in triplicate. 2.3 Product Evolution A second series of titrations (n=19) were conducted to monitor reaction progress and product evolution at pH 6.0, 7.0, and 8.0. Reactions were terminated at no fewer than six varying time points for each pH as shown in Table 1. Time points were selected based on the shape of the titration curves established in the aforementioned experiment.
Table 1: Time points for phase evolution study. Rounded to nearest hour, except if under 1h. pH Time points (h) 6
0.8, 2, 3, 4, 5, 6, 15
7
0.7, 4, 9, 13, 17, 29
8
4, 9, 21, 39, 51, 80
6
2.4 Calcium Phosphoserine Phase Synthesis A pH-stat titration of calcium hydroxide, 1.0 mM Ca(OH)2 with 0.15 M pSer titrant was conducted at pH 8, under N2 atmosphere at 37ᵒC. The goal of this titration was to synthesize the pure coordination compound of calcium phosphoserine (CPS) according to the following chemical reaction. Ca(OH)2 + C3H8NO6P [Ca(C3H6NO6P)(H2O)] + H2O
2.5 Product Characterization The precipitated product from each titration and/or incubation was filtered, rinsed with DI water, and air dried prior to characterization by X-ray diffraction (XRD), FT-IR, and SEM. XRD (Rigaku 007, 40kV/300mA, 2Ө: 10-60ᵒ, step size: 0.02ᵒ, dwell time: 5 s) was performed on each product with an internal standard of corundum (approximately 10 wt.%). Each diffraction pattern was tested for the presence of calcium phosphate phases as well as any other matched phases identified from the Crystallography Open Database (COD-2018). Jade (MDI) Whole Pattern Fitting (WPF) analysis was applied on matched phases using exact corundum values, and the quantified results of each set of experimental trials were averaged to determine mass percent (%) composition. ACP content was determined by the software based on percentage of unmatched and non-crystalline phases. FT-IR spectroscopy (Perkin Elmer Spectrum 100) was used as a secondary qualitative validation of phase composition to corroborate quantitative XRD results. Powder samples were prepared using FT-IR grade potassium bromide (KBr). Approximately 1 % (w/w) of sample was mixed with 99% KBr with an agate mortar and pestle and consolidated into pellets using a stainless steel 7 mm die kit, and a table-
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top hand press (International Crystal Laboratories). Each test included 64 scans (4000-400 cm-1). Characteristic functional groups were identified based on reported absorbance ranges ( Table 2) for similar organic and inorganic compounds. [29-31] Table 2: Relevant FT-IR characteristic group frequencies Functional Group OH st H2O Amines N-H st NH2 δ Carboxylic Acids COO-H st C=O st Phosphates v3PO4 v1PO4 v4PO4 v2PO4
Absorption Range (cm-1) 3200-3650 3000-3700 3300-3500 1550-1650 2500-3550 1650-1800 1000-1150 ~950 500-620 400-470
Scanning electron microscopy (SEM) was also used as an aid for phase identification for the phase evolution study. Dry samples were loaded onto carbon tape and sputter coated (Anatech, Hummer V Sputter Coater) with gold, for 2 min at 15 mA and ~90 mTorr, resulting in a coating thickness of about 10 nm. Imaging was performed using a FEI Scios2 SEM, with the following conditions: HV 5.0 kV, WD ≤ 7 mm, and an ET (mixed secondary and backscatter) detector. Crystal structure and morphology, as well as phase interactions were visualized. 3. Results 3.1 Titration Parameters Figure 3 shows the titration reaction curves, monitoring pSer consumption as a function of time, at pH values of 6.0, 7.0, and 8.0. The consumption was normalized to 1 mol of pure TTCP. The three reaction
8
trials for each pH value were all consistent and reproducible, and each show a characteristic double logistic curve.
Figure 3: Titration curves for pH comparison of pSer and TTCP reaction. Y-axis normalized to 1 mol TTCP.
Titration parameters were calculated and are summarized in Error! Reference source not found.. At the beginning of each titration, the initial dissolution of some TTCP caused an immediate increase in pH, and consequently, a bulk addition of titrant was observed until the preset pH was reached. This initial consumption of titrant was omitted from the rate calculations but included in the total consumption. The consumption and time to reach the desired pH are noted as Ci and ti, respectively. For titration curves with observed inflections, the rate of titration for each region of the graph was calculated individually. The maximum rate for the first part of the reaction (R1) was calculated from the first consistent slope after ti. The second maximum rate of consumption (R2), was calculated from the second increase in consumption after the middle plateau region. The minimum (Rmin) rate of titration for the plateau region was also calculated. The corresponding times for each of these rate calculations are 9
noted below the rate. Completion time for the reaction (tf) was determined as the initial time at which the titrant consumption reached its maximum (Cf) and remained stable for at least 1 h. Table 3: Reaction parameters for pH comparison titrations.
pH 6
Parameters C (mol) R (mol/h) Time (h)
7
C (mol) R (mol/h) Time (h)
8
Ci 0.54 0.02 0.19 0.02 0.08 0.04 0.06 0.04
C (mol) R (mol/h)
-
Time (h)
-
R1 0.28 0.02 0.29 0.02 0.10 0.01 0.51 0.02 0.04 0.00 1.00 0.26
R2 0.18 0.01 5.26 0.73 0.04 0.01 12.07 1.37 0.01 0.00 32.73 8.71
Rmin 0.03 0.01 0.92 0.08 0.02 0.00 7.71 0.60 0.004 0.00 15.87 2.29
Cf 1.29 0.02 11.79 1.12 0.85 0.01 33.52 2.02 0.68 0.03 58.40 1.28
From Error! Reference source not found., it can be seen that Cf, R1, and R2 are inversely related to pH. The product of pH 6 consumed nearly twice as much pSer as that of pH 8, and the reaction was complete in 80% less time. The maximum reaction rates, R1 and R2 (corresponding to the initial and second growth curves), decrease with pH, and in all cases R1 is greater than R2. The peak rate of the second growth curve (R2) is reached after 72% total consumption for pH 6, 68% for pH 7, and 60% for pH 8.
3.2 Product Characterization Figure 3 summarizes the average XRD patterns (from three trials) for each pH at t=0 d and t=7 d. The patterns confirmed the presence of two primary crystalline phase products, hydroxyapatite (HA)
10
and calcium phosphoserine monohydrate complex (CPS, Figure 4), in addition to the amorphous content. The measured α-TCP, which represented only 15 wt% of the starting material, was completely consumed at the one week time point for all pH values, as can be seen in the diffractograms (Figure 5, dotted drop line). Considering that the reactions of both TTCP and α-TCP with pSer yielded the same two phases after 7 days, the effects of residual α-TCP at t=0 were deemed negligible for phase analysis.
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Figure 5: Average XRD patterns for titrations conducted under pH values of 6, 7, and 8, with incubation time points of 0 and 7 days. CPS marked by solid circle, HA by solid triangle, α-TCP by open circle (and dashed drop line), and corundum internal standard by asterisk. Y-axis is arbitrary units (AU) of intensity; plots are y-offset for clarity.
Figure 4: XRD pattern of titration product of CPS reaction. CPS marked by green lines, Corundum standard peaks marked by blue lines and asterisk.
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The CPS synthesized by the Ca(OH)2 reaction in the present work was determined to be highly crystalline and phase pure, as shown in the diffractogram in Figure 4, with colored lines noting matched phases from the COD database. Qualitative FT-IR analysis (Figure 6) of chemically pure CPS as well as the titration products supports the presence of both HA and CPS, specifically with the phosphate vibrations in the fingerprint region and distinct peaks highlighting the presence of both primary amines and carboxyl groups. The relative intensities of the carboxyl and amine peaks decrease with pH, while the phosphate peaks broaden to closely match the spectrum of HA. Figure 7 shows a representative SEM image of each titration product, including the pure CPS product. Figure 8 summarizes the phase composition (mol %) assuming stoichiometric formulas of the titration products showing CPS as the primary phase at every pH value and time point. Percent crystalline product is the sum of CPS and HA, compared to the amorphous and/or nanocrystalline content represented by ACP.
Figure 6: FT-IR spectra of titration products from pSer and TTCP at pH 6, 7, and 8. CPS and HA shown for reference. Relevant functional group ranges shown per Table 2.
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Figure 7: SEM images of (A) CPS, (B) pH 6, (C) pH 7, and (D) pH 8 titration products.
Figure 8: Phase composition (mol%) of titration reaction products for pH 6, 7, and 8 at time points t=0d and t=7d.
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3.4 Product Evolution The phase evolution of the titration reaction products at pH 6, 7 and 8 were also analyzed by XRD and whole pattern fitting, the results of which (moles solid products) are summarized in Figure 9, overlaid with each titration curve. At the completion of the initial growth curve, the main product formed is ACP, with a small amount of HA. At every pH, detection of CPS corresponds with the second growth curve with an initially high rate of formation at that time point. In contrast, the crystallization and precipitation of HA is much more gradual. The time series of SEM images graphically depicts this phase evolution as a function of time. Figure 10 shows an initial formation of ACP (beaded structure), followed by the precipitation and growth of the monoclinic blooming crystals of CPS, as well as the foam-like characteristic growth of HA. Both ACP and HA have been previously confirmed to have these structural features in the presence of organic molecules. [23, 32-34]
15
Figure 9: Phase evolution (by moles) of titration product composition at pH 6, 7 and 8. Product composition based on total solid retrieved at each time point less any unreacted components. Theoretical stoichiometry was assumed for molar conversion.
16
Figure 10: SEM images of phase evolution of pSer titration at pH 6. Phases are labeled as ACP (A), CPS (C) and HA (H). 4. Discussion 4.1 Titration Reactions It is well established that HA is the most common CaP cement product due to its thermodynamic stability in the pH range of 6-8. TTCP has been commonly used in numerous traditional calcium phosphate cements. Most of these cements are composed of purely inorganic phases, usually forming HA or brushite[3]. The results of the titration experiments in this study are consistent with those of a similar study by Chow et al. which examined the effects of pH on reaction kinetics and product 17
composition of a reaction between TTCP and phosphoric acid.[28] In the pH range relevant to this study (pH 7 and 8), they found that the reaction kinetics were mostly limited by TTCP dissolution, because the solution was supersaturated with HA and undersaturated with TTCP. Chow et al. also reported affects of particle size on reaction rate, concluding that smaller TTCP particles accelerated the overall rate. In the present study, the use of a TTCP mixture containing α-TCP and ACP, reduced the relative rate of the reaction. The use of pure TTCP would have increased the pH of the overall reaction due to it high solubility and alkalinity and would have required greater volumes of pSer at an accelerated dispensing rate to maintain the set pH. The heterogeneous mixture of TTCP and α-TCP allowed for an extended reaction time due to α-TCP’s slower dissolution at the given pH range.[35] Unlike the Chow et al. study with phosphoric acid, which produced a single logarithmic growth curve, the present study resulted in a characteristic growth curve with two inflections, suggesting that pSer plays a dual role and results in two distinct reaction pathways. In contrast to phosphoric acid, pSer has the reactivity of both a Bronsted-Lowry acid as well as a Lewis base. This dual reactivity is believed to contribute to the double logisitic curve as well as the formation of CPS, the predominant phase in this adhesive cement system. CPS was first reported by Suga and Okabe in 1996, when they synthesized the compound using an evaporation method from a 5:1 molar mixture of pSer and CaCl2 ∙ 2H2O at pH 7.[36] Their characterization of CPS, which they referred to by its coordinate compound name, aqua (L-O-serine phosphato) calcium (II), was limited to the identification of its crystal structure. Interestingly, in CPS, pSer does not act as a chelating ligand (i.e. one ligand donating multiple electron pairs to a single metal ion) as was previously theorized in the literature about its Ca2+-binding mechanism.[24, 37-41] Instead, each pSer molecule bonds to five distinct calcium ions (each in a monodentate manner), serving as a bridging ligand.[42] Although not previously identified as such, CPS meets the criteria for being a coordination network, as modeled in Figure 11. 18
Despite its chemical identification 20 years ago, the potential link between CPS and biological mineralization has not yet been explored. A study by Wu et. al. confirmed the presence of general calcium-organic phosphate complexes in early stages of bone mineralization, suggesting a potential role for molecules like CPS in the initiation of bone calcification. [43] A recent study by Kirillova et al. reported the mechanical and biological properties of the TTCP-pSer cement system, however, the chemical composition of the cement was not characterized.
They found that in addition to its
osteoconductive properties the cement product also exhibited adhesive properties, suggesting the presence of CPS. [8] Another recent publication sought to determine the molecular basis of adhesion for the reaction products of pSer and α-TCP [9]. Despite the presence of CPS in their XRD data, it was not identified nor discussed, rather several of its peaks were erroneously attributed to α-TCP. Based on the results of the present study and the identification of CPS as the main component of the TTCP-pSer system, it is likely that the self-assembly of the chemically continuous structure of its coordination network gives rise to the unique physical properties and adhesive/cohesive interactions reported by both Kirillova et al. and Pujari-Palmer et al.
Figure 11: Three dimensional model of CPS crystal structure. (CrystalMaker Software)
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4.2 Product Evolution The product evolution experiments give further insight into the progress of the reaction and formation of the individual phases in this cement system. This study (Figures 9 and 10) suggests that the initial growth curve is representative of TTCP dissolution, which leads to a rapid increase in alkalinity. In the absence of pSer, and the use of phosphoric acid as a titrant, Chow et al. documented a single growth curve with TTCP dissolution coinciding with the precipitation of the HA product. However, in this system, it is proposed that the initial asymptote is the result of pSer adsorption onto both TTCP and ACP, which slows dissolution of TTCP. Similar stabilizing effects have been previously reported for calcium phosphates and calcium carbonates in the presence of pSer and other organic molecules.[18-20] For instance, in Figure 10, at the 0.8 h timepoint at pH 6, the TTCP particles are already covered in amorphous/nano-crystalline phases, limiting diffusion. This decreased dissolution as well as the possible inhibition of HA crystallization creates a temporal shift in the classic HA-forming reaction. This hypothesis is supported by the abundance of ACP, which is a known precursor phase for HA; its presence in the product indicates that the formed HA is partially crystalline—broad peaks caused by small, nano-scale crystals. This observation has been also made by others in the literature.[28, 44] The presence of organic entities is also known to produce less crystalline and non-stoichiometric HA.[28, 45, 46] Despite the heterogeneous composition of the starting TTCP mixture, containing α-TCP and ACP, it is expected that in the pH range of 6-8 and at 37C, the two thermodynamically stable phases for the pSer and CaP system are CPS and HA. CaP chemistry has well established the phase diagrams for the Ca-PH2O at various temperatures and pH values.[35] Similarly, an understanding of pSer speciation with respect to pH supports the formation of CPS due to the high abundance of pSerH2- in the pH range of 68.[47]
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Based on evidence from the titration curves and materials characterization, we propose the following reaction pathways for the TTCP-pSer organo-ceramic system in the physiologic pH range:
3Ca4(PO4)2O + 3H2O 3Ca10(PO4)6(OH)2 + 2CA(OH)2 HA Formation
a4(PO4)2O + 3H2O + 4C3H8NO6P 4[Ca(C3H6NO6P)(H2O)] + 2(H3PO4) CPS Formation
These pathways, although shown in parallel, are interdependent due to their common reactant (TTCP) as well as their potentially reactive by-products. For example, Ca(OH)2, produced during HA formation, can also react with pSer to from CPS. Similarly, phosphoric acid, produced during CPS formation, can also react with TTCP to form HA and other CaP phases. The pH of the system can further favor or hinder each pathway based on the solubility of both intermediate and final products. As a biomaterial, this cement system is designed to be mixed and injected for curing in situ. In a physiologically buffered environment, the system becomes increasingly complex due to the potential participation of other ions in the reaction. The fast curing time (as reported by Kirillova et al), may initially hinder the diffusion of many of these ions within the cement, however, over time it is expected that the cement will evolve with the surrounding mineral environment in a similar fashion as biological HA. The local pH of the cement system as it cures in vivo is also expected to vary based on the pSer:TTCP ratio, which might in turn affect the phase composition of the final product. Although HA and CPS are the expected products at physiological pH, a lower pH, for example, might result in brushite or a different coordination network of pSer and calcium ions. The present study provides insight into the 21
unique CPS coordination network which forms under a subset of controlled conditions mimicking this cement system and suggests a more complex family of organo-ceramic phases which likely form under varying pH and ionic conditions. 5. Conclusions The primary purpose of this work was to better understand the reaction kinetics and chemical composition of a novel class of cements which are currently being clinically developed. In contrast to the inorganic components of traditional CaP cement and their potential organic additives, the present study revealed that both TTCP and pSer chemically participate in a set of interdependent reaction pathways that lead to a uniquely adhesive composite cement system. The effects of pSer on the reaction kinetics of HA crystallization is believed to result in the temporal shift observed in the signature double logistic curve of the reaction profile for this system. It was determined that pH has a significant effect on the reaction rate of TTCP-pSer, however, the system’s products are robust within the pH range of 6 and 8. Phosphoserine’s participation in this reaction is a result of its dual functionality as both a Brønsted-Lowry acid and Lewis base. Furthermore, this work revealed the previously unidentified presence of the coordination network, CPS, in an apatitic cement system. This crystalline compound was found to be the predominant phase for this system at all tested pH values. The fundamental findings presented here, better prepare our group for future functional mechanical testing and biological assessment of this system. The unique multiphasic composition of this system may provide insight into the complexities of bone mineralization as well as set the stage for further development CPS-based biomaterials and cements with functional physical properties.
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F.P.K. and C.S.L performed the titrations. F.P.K., C.S.L., and D.W.VC. designed the experiments. F.P.K. wrote the paper. All authors analyzed data and edited paper. We thank Dr. Laurence C. Chow for useful discussions about calcium phosphate chemistry. This research has been supported by a Small Business Technology Transfer grant from the National Institute on Aging of the U.S. National Institutes of Health, Award Number R41AG060881. Declaration of interests None
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Figure Captions List
Figure 1: TTCP mixture compared to reference patterns for pure TTCP, α-TCP, and HA. Figure 2: Schematic of pH-stat titration. Figure 3: Titration curves for pH comparison of pSer and TTCP reaction. Y-axis normalized to 1 mol TTCP. Figure 4: Average XRD patterns for titrations conducted under pH values of 6, 7, and 8, with incubation time points of 0 and 7 days. CPS marked by solid circle, HA by solid triangle, α-TCP by open circle (and dashed drop line), and corundum internal standard by asterisk. Y-axis is arbitrary units (AU) of intensity; plots are y-offset for clarity. Figure 5: XRD pattern of titration product of CPS reaction. CPS marked by green lines, Corundum standard peaks marked by blue lines and asterisk. Figure 6: FT-IR spectra of titration products from pSer and TTCP at pH 6, 7, and 8. Figure 7: SEM images of (A) CPS, (B) pH 6, (C) pH 7, and (D) pH 8 titration products. Figure 8: Phase composition (mol%) of titration reaction products for Figure 9: Phase evolution (by moles) of titration product composition at pH 6, 7 and 8. Product composition based on total solid retrieved at each time point less any unreacted components. Theoretical stoichiometry was assumed for molar conversion. Figure 10: SEM images of phase evolution of pSer titration at pH 6. Phases are labeled as ACP (A), CPS (C) and HA (H). Figure 11: Three dimensional model of CPS crystal structure. (CrystalMaker Software)
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Graphical Abstract
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