Modulation of Hydroxyapatite Nanocrystal Size and Shape by

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DOI: 10.1021/cg900750z

Modulation of Hydroxyapatite Nanocrystal Size and Shape by Polyelectrolytic Peptides

2009, Vol. 9 5220–5226

Jared J. Diegmueller, Xingguo Cheng, and Ozan Akkus* Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907 Received July 2, 2009; Revised Manuscript Received October 2, 2009

ABSTRACT: Interactions of synthetic peptides containing charged amino acids with hydroxyapatite (HAP) have been used as models of non-collagenous proteins’ interaction with bone’s minerals. While there is some knowledge of the affinities of these peptides with HAP, the effects of the type of charged peptide, peptidic molecular weight, and concentration on the crystal morphology have not been systematically investigated. Such knowledge could be useful for identifying the peptides ideal for in vitro or in vivo administration to customize crystal growth or for coating of implant surfaces for improving anchorage to neighboring bone tissue. This study used charged peptides of arginine (Arg), aspartate (Asp), glutamate (Glu), and lysine (Lys) to influence the HAP morphology. A systematic approach evaluated the polyelectrolytic peptides’ affinity for HAP as well as their effects on mineral nucleation/growth kinetics and crystal morphology. Negatively charged polymers (Asp, Glu) had greater affinity for hydroxyapatite than positively charged polymers (Arg, Lys). Polymers of higher molecular weight (HMW) had greater affinity for HAP than their lower molecular weight (LMW) counterparts. Negatively charged polymers at lower concentrations created more mineral mass at greater crystallinity. Therefore, polymers of greater affinity have the most effect on crystal growth kinetics. However, these effects are inhibited at higher concentrations. Generally, the addition of any polymer decreased the dimensions of the HAP crystals formed with respect to control. Poly-L-Asp LMW, poly-L-Glu LMW, and polyL-Lys HMW were found to significantly affect crystal morphology increasing the aspect ratio in comparison to the control. In conclusion, these results suggest that polyelectrolytic peptides may be useful in vitro or in vivo administration to customize size and shape of HAP nanocrystals or for coating of implant surfaces for modulation of implant attachment to neighboring bone tissue.

Introduction The interaction of macromolecules with calcium phosphate plays an important role in biomineralization of bone and teeth, pathological mineralization such as atherosclerosis and dental calculus, and industrial applications.1,2 Noncollagenous proteins present in mineralized tissues contain a large number of charged amino acid residues such as arginine (Arg), lysine (Lys), aspartic acid (Asp), and glutamic acid (Glu). Bone sialoproteins (BSP) and osteopontin each contain ArgGly-Asp motifs which have been found to play multiple roles in the biomineralization process such as binding hydroxyapatite (HAP), attaching to cell-surface integrins, and cell recognition.3-7 The gradual discovery of the role of charged amino acid-containing proteins in the biomineralization process has aroused extensive interest in the use of their analogsynthetic polyelectrolytic peptides to determine their effects on mineral crystals. Understanding the role of charged peptides in nucleation and growth of crystals is critical to mimicking the biomineralization process toward synthesis of novel biomaterials.8-12 In this context, the influence of poly-L-Lys, poly-L-Glu, and poly-L-Asp on crystallization of calcium phosphate has been extensively studied.13-20 An emerging approach for identification of solid binding organic materials is based on bioinformatic analysis of phage display libraries.21-25 These efforts have been successfully applied to identify peptides with high and low affinities to hydroxyapatite.21 *To whom correspondence should be addressed. Address: Weldon School of Biomedical Engineering Purdue University 206 S. Martin Jischke Drive West Lafayette, IN 47907-2032. Tel.: (765) 496-7517. Fax: (765) 496-1912. E-mail: [email protected]. pubs.acs.org/crystal

Published on Web 11/12/2009

It is known that binding of a synthetic polymer or peptide on colloidal particles can affect the dissolution of mineral, change the growth kinetics of the precipitated crystal, or initiate nucleation of crystals. Therefore, peptide binding could be used to modulate bone crystal size and shape through peptide-bone crystal interaction, and it has long been suggested that these agents may affect the nanobiomechanical properties of bone.26 Composite mechanics models of bone simulating the effect of nanocrystal morphology predict that stiffness of bone increases with increasing length/width ratio.27 Furthermore, there is some experimental evidence that increasing crystal maturity (reflecting larger crystallites with fewer imperfections) is a correlate of increasing bone strength.28 Therefore, modulation of crystal size and shape by using charged charged peptides has the long-term potential for controlling strength of bone tissue as well as for designing artificial calcified biomaterials.29 Previous studies have investigated charged peptides individually or in subgroups;17,30,31 however, there is a lack of understanding their relative standing in terms of their effects on HAP formation. Furthermore, the effects of the molecular weights and concentrations of charged peptides on crystal morphology are not fully understood. The first aim of the current study is to systematically evaluate the affinity between HAP and poly-L-Arg, poly-L-Asp, poly-L-Glu, and poly-L-Lys of different molecular weight through adsorption studies. A ranking of such affinities is essential for identifying the peptides ideal for in vitro or in vivo administration to customize crystal growth or for controlling the dissolution and growth of crystal particles or films. The second aim of this work is to assess these polyelectrolytic peptides as biomimetic analogs of noncollagenous r 2009 American Chemical Society

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Table 1. Polypeptide Structures at Physiological Conditions and the Molecular Weights Examined during Affinity Experiments

proteins in terms of influencing the size and shape of hydroxyapatite crystals synthesized in vitro. The effect of polyelectrolytic peptides on the induction, amorphous phase formation, crystal growth kinetics of the hydroxyapatite is systematically examined. On the basis of the knowledge from the two aims, a subgroup charged peptides were screened to characterize their ability to modulate the size and shape of HAP nanocrystals as the final aim. Materials and Methods The current study evaluated four polyelectrolytic peptides (Sigma), two of which were positively charged (poly-L-arginine and poly-L-lysine) and two were negatively charged (poly-L-aspartic acid and poly-L-glutamic acid). Each peptide was evaluated at two molecular weights (Table 1). Affinity of Polyelectrolytic Peptides to Hydroxyapatites. Peptides were mixed with HAP crystals, and the amount of unbound peptide in the solution was measured. Hydroxyapatite (Sigma-Aldrich, St. Louis, MO) crystals were added to 35 mL HAP saturated solutions at an amount of 25 or 50 mg.31 The HAP saturated solution (0.07 mM CaCl2, 0.042 mM KH2PO4, 0.15 M NaCl in ultrapure water) was used to avoid the dissolution of the added HAP crystals. The saturated solution was added with 0 mg/L (control) or 50 mg/L of each peptide and then HAP crystals were added. The solutions were incubated and rotated via a rotator (Rugged Rotator 099A, Glas-Col, Terre Haute, IN) at 25 rpm for 4 h in a 37 °C incubator. The incubated samples were centrifuged at 2500 rpm for 10 min (Allegra X-12R, Beckman Coulter, Fullerton, CA) and the supernatant was collected. Centrifuged HAP crystals were rinsed with 20 mL HAP saturated solution and recentrifuged to add the second supernatant to the first collection. The absorption of each solution was measured using an ultraviolet-visible (UV-vis) light spectrometer (SpectraMax M5, Molecular Devices, Sunnyvale, CA) at 200 nm to measure the peptide concentration remaining in solution after binding.17 HAP saturated solutions with known amount of peptides were used to establish UV-vis calibration curves for concentration measurements. Samples were divided into 18 aliquots and measured 9 times per aliquot to obtain the average unbound peptide concentration. Unbound peptide weight was subtracted

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from the originally added amount to determine the amount of peptide bound to the HAP. Crystal Growth Kinetics. Nucleation and growth of crystals were assessed by turbidity measurements. All CaCl2 and KH2PO4 solutions used were prepared using a tris-buffered saline solution which contains 42 mM Tris-HCl, 8 mM Tris-Base, and 150 mM NaCl (pH 7.4 at 37 °C). The turbidity of each solution was measured by kinetic profiling on a UV-vis spectrophotometer (SpectraMax M5, Molecular Devices Corp., Sunnyvale, CA). Volumes of 100 μL of both calcium and phosphate solutions for each different concentration level were dispensed into wells of a 96-well plate. The plates were placed into the UV-vis spectrophotometer to obtain absorption readings at 313 nm, every 20 s for up to 8 h, and at 37 °C. Each group was evaluated in replicates of four. All the solutions and the 96-well plates were preheated to 37 °C and then 100 μL of calcium and phosphate solutions were added to the plate. Optimal concentrations for calcium and phosphate solutions were determined prior to kinetic measurements. Optimal concentration was that which offered substantial enough delay before nucleation to determine whether a charged peptide increased or decreased the induction time of nucleation; however, it still allowed the entire growth phase to occur. This growth phase was verified by the attainment of the steady state during the 8-h turbidity measurement. The optimal calcium and phosphate concentrations which fulfilled these conditions were found to be 7.5 mM CaCl2/3.5 mM KH2PO4 and were utilized for the ensuing experiments. The peptides used previously in the affinity studies (Table 1) were examined at the following concentrations: 2.5 mg/L, 7.5 mg/L, and 25 mg/L as well as including one group containing no peptides as control. At 7.5 mM CaCl2/3.5 mM KH2PO4 concentrations, the kinetic growth profiles exhibited three phases, the induction, amorphous growth, and crystalline growth (Figure 2a, Curve II). There were distinct transitions in the absorption rates between each phase that allowed their identification. The durations of each phase were obtained from the kinetic absorption profiles. Also, the amount of crystal formation at a given phase was inferred from the absorption amount at the given phase. The induction period was defined as the duration it takes for the crystals to precipitate after initial mixing of the two solutions. Raman Spectroscopy. The crystal kinetics of the solutions was arrested during amorphous and crystalline growth phases, an aliquot was taken from the solution, the crystals were filtered under a vacuum, and dried at ambient conditions. The Raman spectra of dried crystals were taken by a Raman microscope (Labram HR800, Horiba Jobin Yvon, Edison NJ) to obtain their crystallinity. Raman-based crystallinity cumulatively reflects both crystal size and crystal perfection such that an increase in both measures is manifested as an increased crystallinity variable.28 The system consists of a laser source at 660 nm, and measurements were performed using a 600 lines/mm grating which provided a wavenumber resolution of 1.25 pixels/cm-1. The Raman wavenumber shift measured by the system was calibrated using the known 520.7 cm-1 peak of a Si wafer. Crystallinity of precipitates was inferred from the full width at half-maximum (fwhm) of the phosphate symmetric stretch peak (∼960 cm-1) such that the inverse of fwhm is positively correlated to crystallinity. Batch Experiment to Determine Polyelectrolyte Effects on Crystal Size and Geometry. The results of the turbidity and affinity experiments were used to narrow down the choice of peptides, the molecular weights, and their concentrations for assessing their effects on crystal morphology and size. Each of the poly amino acids (Arg, Asp, Glu, and Lys) was evaluated at the lower concentration of 2.5 mg/L, and poly aspartic acid was further evaluated at the higher concentration of 25 mg/L. These offer a breadth of the samples and those that significantly increased the absorption results of the turbidity measurements (i.e., maximized crystal precipitation). As the molecular weight of poly arginine was later found to have little effect in terms of the UV absorption, only the lower molecular weight sample was examined. A sample containing no poly amino acid was used as control. Batch precipitations were conducted by mixing 500 mL each of calcium and phosphate solutions (15 mM CaCl2 and 7 mM KH2PO4, respectively). These samples were incubated at 37 °C for 8 h while being shaken at 25 rpm

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(Forma Orbital Shaker 480, Thermo Electron Corporation, Waltham, MA), and then collected by filtration using an 11 μm cellulose filter as the particles coalesce to form large aggregates. Any samples whose filtered solution still showed cloudiness were further filtered using a 0.22 μm cellulose filter and added to the previous collection. A high degree of aggregation was observed in filtered solutions and a dialysis process32 was carried out to isolate nanocrystals from aggregates prior to transmission electron microscopy. In isolating, samples were resuspended in the HAP saturated solution used in affinity experiments and placed into dialysis bags. The dialysis bags with a molecular weight cutoff of 3500 kDa (Fisherbrand #21-15210, Fisher Scientific, Pittsburgh, PA) were placed in 1 L of Milli-Q water for a 24 h duration during which the 1 L of water was replaced once. The dialyzed hydroxyapatite crystals were then evaluated using a FEI/Phillips CM-10 TEM. TEM sample preparation included depositing ∼8 μL of each sample directly onto a Formvar/ carbon coated 400 mesh TEM grid, allowing the crystal to settle, and staining with phosphotungstic acid (PTA) at a pH ∼7. Using Scion Image (Scion Corporation, Fredrick, MD), the nanocrystals perimeters were traced and crystal dimensions were determined using the scale measurements, evaluating longest and shortest dimensions of platelets (reported as major and minor axis, respectively). The aspect ratio of crystals was determined by dividing the major axis by the minor axis. Twenty-three crystals were observed as such for each treatment group to report the mean crystal dimensions. Many individual nanocrystals with identifiable boundaries were available following dialysis treatment. There were also groups of nanocrystallites which aggregated. If the contiguity of more than two crystals and the extent of aggregation hindered the identification of an individual crystallite, such crystals were not measured. Therefore, TEM characterization was performed on crystals which stood alone with unambiguous boundaries. Statistics. Statistical analysis for the affinity and crystal growth kinetics was performed using Minitab (Minitab Inc., College Station, PA). A general linear model analysis of variance was performed to test for the effects of peptide type, HAP amount, peptide concentration, and peptide molecular weight on the bound peptide mass or the turbidity profile at different stages. Tukey’s post hoc test was used for pairwise comparisons. The significance of differences between the dimensions of nanocrystals were assessed by a Kruskal-Wallis test (Minitab Inc., College Station, PA) and posthoc differences of polyelectrolyte treated groups and the untreated controls were assessed by Dunn’s test. The level of significance was set at p < 0.05 and marginal significance reported for 0.05 g p g 0.10. The significance of the relations between kinetic growth parameters, namely, the absorption and induction time were assessed by linear regression analysis.

Results The affinities of peptides of similar molecular weight for HAP are ranked as follows: poly-L-Asp > poly-L-Glu > poly-L-Lys > poly-L-Arg (Figure 1). Negatively charged peptides bind to HAP more strongly than the positively charged peptides. For example, for 25 mg of HAP, poly-LGlu LMW bound more than four times the amount of peptide to the HAP surface than poly-L-Lys LMW. Among the negatively charged peptides, poly-L-Asp binds to HAP 5% to 43% more strongly than poly-L-Glu, depending on MW and concentration. Anionic peptides with higher molecular weight bind more strongly to HAP than the ones with lower molecular weight (Figure 1). This effect of molecular weight on peptide affinity to HAP is the most pronounced for poly-L-Glu. In the positively charged peptides, it was observed that poly-L-Arg of higher molecular weight binds at three times greater amounts to HAP than poly-L-Arg of low molecular weight. In evaluating the optimal concentrations of calcium and phosphate for turbidity profiles, lower concentrations were found to have too long a delay (greater than 8 h) before

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Figure 1. Amount of peptide, in milligrams, bound to different amounts of HAP. The negatively charged peptides (Asp, Glu) bound significantly more to HAP than the positively charged peptides (Arg, Lys). The higher molecular weight of each peptide also significantly bound more to HAP than the lower molecular weight of the peptide for all peptides except Lys.

nucleation, while greater concentrations induced instant nucleation (Figure 2A). The concentration of 7.5 mM CaCl2/3.5 mM KH2PO4 offered a reasonable delay of about 15 min prior to nucleation as well as the turbidity profile reached the steady state within 8 h. Crystals which were collected during the amorphous phase had broader phosphate symmetric stretch peak width and lower wavenumber than the 8 h sample (Figure 2B). The effect of the different poly amino acids on the induction time, or the delay before crystal growth, is shown in Figure 3A. Poly aspartic acid in the low molecular weight form induces the greatest delay on induction time. This delay is directly related to the concentration such that the induction time increases with increasing concentration. Poly lysine of low molecular weight significantly increased the induction time, although to a much lesser extent than poly aspartic acid. Poly aspartic acid LMW at the highest concentration significantly increased the duration of amorphous growth phase (Figure 3B). The addition of polypeptides generally increased the UV absorption (i.e., amount of crystal formation) during the amorphous growth phase (Figure 4A). An increasing concentration of the peptide translated to a reduction in the amount of crystal growth during the amorphous phase for the negatively charged peptides whereas this concentration effect was absent in the positively charged peptides. There was not a clear trend in terms of the effects of molecular weight, except that HMW poly-L-Lys led to a greater amount of crystal formation during the amorphous growth phase. The lower concentrations of the negatively charged peptides significantly increased the total absorption during the crystalline growth period (Figure 4B); however, the effect subsided at increasing concentrations. Positively charged peptides did not affect the absorption during crystalline phase with the sole exception of poly-L-Arg HMW at 25 mg/L concentration which reduced the reduced the amount of growth with respect to controls. It was observed that the sooner the induction took place, the more crystals were grown during the amorphous period (R2 = 0.41) and the less crystals were grown during the crystalline period (R2 = 0.41).

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Figure 2. (A) Turbidity measurement of solutions after mixing different concentrations of CaCl2 and KH2PO4 without any polymer addition (37 °C, pH = 7.4 tris buffer). (I) 9 mM CaCl2/4.2 mM KH2PO4, (II) 7.5 mM CaCl2/3.5 mM KH2PO4, (III) 4.5 mM CaCl2/ 2.1 mM KH2PO4. The induction phase duration (DI), amorphous growth phase duration (DA), and crystalline growth phase duration (DC) are highlighted for the curve in II. (B) The Raman spectra of samples collected in the amorphous growth (dark curve) and crystalline growth phases (light curve).

The major and minor axes of crystal dimensions were significantly reduced when they were synthesized in the presence of polyelectrolytes (Table 2). The shape of the crystals became more elongated (i.e., greater aspect ratio) under the effect of negatively charged poly electrolytes. It was observed that negatively charged peptides lead to smaller crystals than positively charged ones (Figure 5 and Table 2). Discussion This study used charged peptides to influence the HAP morphology. A systematic approach evaluated the polyelectrolytic peptides’ affinity for HAP as well as their effects on mineral nucleation/growth kinetics and crystal morphology. Negatively charged polymers (Asp, Glu) had greater affinity for hydroxyapatite than positively charged polymers (Arg, Lys). Polymers of higher molecular weight (HMW) had greater affinity for HAP than their lower molecular weight (LMW) counterparts. Negatively charged polymers at lower concentrations create more mineral mass as an increased absorption is experienced during HAP crystal formation and growth. Therefore, polymers of greater affinity have the greatest effect on crystal kinetics. However, these effects are inhibited at higher concentrations. Generally, the addition of any polymer decreased the dimensions of the HAP crystals formed with respect to control. Poly-L-Asp LMW, poly-LGlu LMW, and poly-L-Lys HMW were found to significantly affect crystal morphology by increasing the aspect ratio in comparison to the control. Extensive aggregation of crystals was observed in all the groups, including those treated with peptides, indicating that peptides used in this study do not counter aggregation. A

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Figure 3. The effect of charged peptides on different molecular weights and varying concentrations on (A) the induction time prior to nucleation. (B) The duration of the amorphous growth phase for the turbidity profiles (“*” indicates significant difference from control).

dialysis scheme was employed for isolation of crystallites from the aggregates. This approach was employed by Zhang et al. to grow fluoride substituted hydroxyapatite nanocrystals without aggregation.32 In the current study, this approach was observed to disaggregate precipitates prepared a priori to nanocrystals. It is likely that excess charges and loosely bound ions which are aggregating HA nanoplatelets are removed during the dialysis process, separating the crystals. There is the concern that nanocrystals may have dissolved and changed dimensions during the dialysis process. Saturated HAP solution was employed during dialysis. Also aliquots were taken from the dialysis solution for up to 160 h and the pH of aliquots stayed within 7.3 þ/- 0.3 during the period. Given that others have grown crystals under such conditions and that the pH did not change notably, it is likely that the dialysis treatment did not affect crystal dimensions substantially. This assertion remains to be confirmed by size analysis of crystals from aliquots collected as different time points. Therefore, some reduction in crystal dimensions may have taken place during dialysis. However, such affects may be comparable between the treatment groups all of which were dialyzed under similar conditions. Previous literature assessed charged peptides’ effects on HAP growth at a single molecular weight13,33-35 and generally at single concentration. Furthermore, previous studies entailed nonphysiological pH values and temperatures, whereas this study focused on the interaction of polymer and hydroxyapatite under physiological pH and temperature conditions. While the pH was not actively controlled during the kinetic growth assays, the starting pH for all solutions was buffered at 7.4; therefore, potential changes in pH by addition of peptides were offset. The final pH of the solutions was

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Figure 5. TEM images of (A) poly-L-Asp LMW 2.5 mg/L and (B) poly-L-Lys HMW 2.5 mg/L.

Figure 4. The effect of charged polypeptides of different molecular weights and varying concentrations on (A) absorption during the amorphous growth phase of the turbidity profile, and, (B) absorption during the crystalline growth phase of the turbidity profile (& and ˆ indicate significantly smaller and greater from the control, respectively).

always greater than 7.0. The current study implemented a systematic approach by varying the charge, molecular weight, and concentrations to determine which combination affects crystal size and shape. The reported affinity rankings here agree with the ranking of polyelectrolytes in terms of their ability to induce or inhibit crystal growth as reported by others.30 Therefore, binding affinity between a peptide and HAP is the key contributor to both induction of mineral growth and inhibition of secondary nucleation. Within the negatively charged peptides, we observed the binding affinity of poly-L-Asp to be greater than poly-L-Glu which is consistent with earlier reports.31 The higher affinity of negatively charged polymers to HAP crystals suggests that they may associate with the lattice spaces for phosphates or hydroxyl groups more readily. It can also be speculated that the positively charged polymers may associate with calcium positions which hold a lower percentage of lattice spaces than the negatively charged ions, thereby, reducing the affinity of positively charged polymers to HAP. The novel contribution of the current work is that the molecular weight of the polyelectrolytic peptide plays a crucial role in determining the degree of interaction with HAP as the larger molecular weight peptides bind to peptides more readily. There are various mechanisms which may explain this observation. The lengths of higher molecular weight peptides

in the extended and form is about 40 nm and about twice longer than the lower molecular weight counterparts. The greater length would result in a greater number of attachment points and would potentially decrease the dissociation of the peptide from the crystal surface. Another possible mechanism favoring stronger attachment between higher molecular weight peptides and HAP crystals could be steric attraction. These mechanisms need to be further investigated in the future. Crystal growth kinetics experiments indicated that increasing the induction time and decreasing the duration of amorphous growth phase led to a greater amount of mineral produced during the crystalline growth phase. In general, the negatively charged poly amino acids, aspartic acid and glutamic acid, at lower concentrations increased crystal production. Regardless of charge type, almost all peptides increased the amount of crystal production during the amorphous nucleation phase; however, only the negatively charged poly aspartic acid and poly glutamic acid increased the amount of absorption during the crystalline nucleation and growth phase. While the absorption is a reliable measure of the amount of crystal formation, the limitation of the technique is that it cannot explain whether the increase in the amount of crystal is due to increase in number of crystals (i.e., nucleation), increase in the size of them, or the combination of the two. Generally, poly aspartic acid and poly glutamic acid had approximately equal effects throughout the turbidity duration. Also, increasing the concentration of the polymers resulted in less absorption in every stage of crystal formation. This indicates that higher concentrations of the polymers curb crystal nucleation and growth which has been reported by others as well.30 Except for increasing the absorbance during the amorphous growth phase, which all polymers generally did, poly arginine and poly lysine had little effect. The results of this experiment illustrate that the increased affinity attributed to the negatively charged polymers increased the crystal amount measured approximately 30% during the crystalline growth phase and 40% during the total turbidity profile. However, the increased affinity contributed to higher molecular weights did not amount to an increase in crystal amount. Both LMW and HMW samples had approximately comparable effects on the amount of absorption for each of the polyelectrolytic polymers. Therefore, affinity by charge seems to be the only determining factor in influencing the crystal amount, whereas affinity by way of molecular weight does not affect crystal yield.

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Table 2. Schematic Representation of Hydroxyapatite Crystal Morphologya

a The schemas are drawn to scale by using the values listed underneath. The morphological measures are reported as mean ( standard deviation obtained from TEM measurements of 23 individual crystallites in each treatment group. The listed values represent the length along longer axis, the length along shorter axis, and the aspect ratio, respectively.

The addition of every peptide successfully decreased the hydroxyapatite crystal sizes with respect to the control. Poly aspartic acid LMW for both concentrations, poly-L-Glu LMW and poly-L-Lys LMW increased the aspect ratio of the hydroxyapatite crystals and resulted in more elongated crystals. The data in the overall indicate that negatively charged peptides, particularly poly aspartic acid at low molecular weight, decrease the size of hydroxyapatite crystals as well as make them more spindle like. While hydroxyapatite is not representative of mineral observed in bone, it was observed that the crystal dimensions and aspect ratios for peptide treated samples were similar to those reported for bone crystals.8 The absence of electron diffraction analysis of nanocrystals in this study prevents the identification of the mechanisms by which the peptides influence crystal morphology. However, several potential mechanisms can be proposed. The crystal morphology is determined by the growth rates of the crystal on the different surfaces.36 The poly amino acids may bind preferentially with the different crystal surfaces. Generally, the absorption of a molecule onto the crystal surface will inhibit or

decelerate the crystal growth planes in that direction.37 The protein fixed to the crystal surface may additionally act as a site of crystal nucleation causing an increased yield in crystal formation.38 These mechanisms remain to be confirmed with further experiments. Presence of negatively charged residues, such as Asp and Glu, does not serve as the only means to regulate crystal growth. Recent studies have identified peptides using phage display libraries and the peptide with the highest affinity to hydroxyapatite lacked Asp or Glu residues.21 There were differences between the secondary structures and the stabilities of the peptides with the lowest and highest affinities; therefore, molecular conformations were proposed as potential effectors of the interaction, as well as the ability of the peptide to interact with phosphate groups. The peptides with the greatest affinity yielded smaller nanocrystals in our study, whereas the higher affinity peptide in the study by Gungormus et al. generated much larger crystals. Such differences may stem from the charge-based nature of the homopolymeric peptides in this study versus the phage library identified peptides which has discrete primary sequences.

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In conclusion, this study demonstrates that hydroxyapatite crystal morphology can be successfully manipulated using charged peptides. The poly amino acid charge (positive or negative), molecular weight, and concentration directly influence crystal morphologies. This study gives support to the notion that the charged noncollagenous proteins present during different stages of bone development might play critical roles in directing hydroxyapatite crystal formation and growth. Successful alteration of crystal morphology and dimensions by charged peptides may be useful in mitigating impaired bone mechanics of diseased states such as osteoporosis. The charged polymers might also prove to be superior substrate coatings for orthopedic implants that may allow better bone integration into the surfaces of the implant. Acknowledgment. The material presented in this study is based upon work supported by the National Science Foundation under Grant No. 0620061 (OA) under the CAREER program. Authors would like to thank Xuanhao Sun for conducting pH measurements during dialysis.

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