Systematic Assessment of Synthesized Tri-magnesium Phosphate Powders (Amorphous, Semi-crystalline and Crystalline) and Cements for Ceramic Bone Cement Applications

Accepted Manuscript Systematic Assessment of Synthesized Tri-Magnesium Phosphate Powders (Amorphous, Semi-Crystalline and Crystalline) and Cements for Ceramic Bone Cement Applications Nicole Ostrowski, Vidisha Sharma, Abhijit Roy, Prof. Prashant N. Kumta, Ph.D. PII:

S1005-0302(15)00035-3

DOI:

10.1016/j.jmst.2014.12.002

Reference:

JMST 469

To appear in:

Journal of Materials Science & Technology

Received Date: 21 September 2014 Revised Date:

7 December 2014

Accepted Date: 9 December 2014

Please cite this article as: N. Ostrowski, V. Sharma, A. Roy, P.N. Kumta, Systematic Assessment of Synthesized Tri-Magnesium Phosphate Powders (Amorphous, Semi-Crystalline and Crystalline) and Cements for Ceramic Bone Cement Applications, Journal of Materials Science & Technology (2015), doi: 10.1016/j.jmst.2014.12.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Systematic Assessment of Synthesized Tri-Magnesium Phosphate Powders (Amorphous, Semi-Crystalline and Crystalline) and Cements for Ceramic Bone Cement Applications Nicole Ostrowski, Vidisha Sharma, Abhijit Roy, Prashant N. Kumta† Departments of Bioengineering, Chemical and Petroleum Engineering and Mechanical

Materials, University of Pittsburgh, Pittsburgh PA 15261, USA

RI PT

Engineering and Materials Science and the Center of Complex Engineered Multifunctional

[Received 21 September 2014; Received in revised form 7 December 2014; Accepted 9 December 2014]

M AN U

E-mail address: [email protected] (P.N. Kumta).

SC

† Corresponding author. Prof., Ph.D.; Tel.: +1 412 648 0223; Fax: +1 412 624 3699.

Magnesium phosphate cements has come under investigation in recent years for use as an alternative to calcium phosphate cements for bone void repair applications. Evidence indicates that magnesium phosphate cements obtain higher initial strengths after cement reaction and resorption in more clinically appropriate time frames than commercially available calcium phosphate cements.

TE D

In this study, amorphous, partially amorphous and crystalline tri-magnesium phosphate powders were synthesized via an aqueous precipitation reaction with subsequent thermal treatment, and characterized using techniques such as X-ray

EP

diffraction and Fourier transform infrared spectroscopy. These materials were assessed for their functionality in cementing reaction with a 3.0 mol/L, pH 7.0 ammonium phosphate solution, including setting time and pH evolution in

AC C

phosphate buffered saline solution. Results indicated that the amorphous and semi-crystalline tri-magnesium phosphate powders were highly reactive with the setting solution but resulted in mechanically weak cements, while the crystalline tri-magnesium phosphate powder reacted efficiently with the cement solution and were mechanically strong following reaction. X-ray diffraction and scanning electron microscopy analyses indicated significant changes in the phase composition and morphology of the cements following incubation in phosphate buffered saline. These were perceived to be detrimental to the integrity of the amorphous and semi-crystalline tri-magnesium phosphate derived cements but not to those created with crystalline tri-magnesium phosphate. The crystalline

1

ACCEPTED MANUSCRIPT

tri-magnesium phosphate material resulted in the most functional cement as this embodiment displayed the most clinically relevant setting time as well as the highest mechanical resilience and neutral pH during incubation in saline solution rendering them potentially viable as bone void fillers.

Amorphous magnesium phosphate; Tri-magnesium phosphate; Bone cement; Bone regeneration; Struvite

SC

1. Introduction

RI PT

Key words:

Bone substitutes are widely used in patients who require implantation to repair or

M AN U

remodel bone defects. These bone substitutes can range from synthetic materials such as metals and polymers to natural polymeric and biologic materials including allografts and autografts[1,2]. Calcium phosphate based synthetic grafts are an excellent choice of bone replacement systems because these implants mimic the chemistry of the mineralized portion of human bone and have shown excellent biocompatibility[3,4]. Currently, there are calcium phosphate based bone cements clinically available, however these products are less than ideal.

TE D

Optimal bone cements should display high biocompatibility and osteoconductivity, strengths similar to natural bone, resorption rates in line with rapid bone remodeling, and clinically appropriate mix-ability, injectability, and setting times[4–6]. Brushite (CaHPO4) based calcium phosphate bone cements, although readily resorbable, tend to display prohibitively fast setting

EP

rates and compressive strengths lower than natural bone. Hydroxyapatite (Ca5(PO4)3(OH)) based calcium phosphate bone cements are capable of achieving higher strengths, but set very

AC C

slowly and require several years to resorb and allow for bone remodeling[3,7]. Recently, it has been proposed that magnesium phosphate cements may pose a more clinically favorable option to traditional calcium phosphate cements[8]. Preliminary studies have shown that magnesium phosphate based cements display higher strengths, better setting time and faster resorption rates than calcium phosphates, while maintaining a high level of biocompatibility[8–16]. However, in contrast to calcium phosphates, the intensity and depth of study into these cement systems is still limited to only a few phases and remains to a large extent unexplored with inconsistent evaluation. The proposed study is to explore the cementing characteristics of amorphous and crystalline tri-magnesium phosphate (TMP), Mg3(PO4)2 ceramic. Trimagnesium phosphate is a potential bone cement precursor material critical to the

2

ACCEPTED MANUSCRIPT

cementing reaction. It is important to study the precursor reactants as they correlate to the final cement product since the material properties affect reaction kinetics, cement formulation and final product properties. The study of magnesium phosphates for bone cements is a relatively new field of study compared to traditional calcium phosphates. Calcium phosphate bone cements have been exploited to a large extent because the composition closely mimics mineralized bone matrix, In addition to

RI PT

however mechanical strength and resorption rate of these cements are not ideal.

calcium, many other ions such as magnesium, silicon, zinc, potassium and copper play important roles in bone regeneration, from regulating the metabolic processes and osteoblastic differentiation genes to promoting angiogenesis and increasing bone cell adhesion[17,18] .

With

SC

regards to bone formation and dissolution, magnesium has been shown to play a major role in calcification and bone density, mineral metabolism, hydroxyapatite crystal formation and

M AN U

increased bone cell adhesion and stability[16,19–21]. Phosphate is not only a main component of bone growth, but has also been shown to stimulate expression of matrix gla protein which is involved in bone formation[17]. However, high concentrations of any of these ions, including calcium, can significantly impede the viability of cells[22,23]. Recent studies into magnesium phosphates have been conducted due to higher strengths and faster resorption rates of these

MgHPO4· 3H2O,

TE D

cements. Magnesium phosphate cement or ceramic compositions explored to date include MgKPO4·6H2O,

Ca3Mg3(PO4)4[8,24–28].

Mg3(PO4)2·8H2O,

MgNH4PO4·6H2O,

and

The use of amorphous calcium phosphates (ACPs) in bone scaffolds and cements is

EP

known, however the level of understanding regarding the potential of amorphous magnesium phosphates for biomedical applications is still very marginal. ACPs are often regarded as highly

AC C

useful as scaffolds or scaffold precursors because of the highly reactive and soluble nature of the ACP[29]. ACP is a critical part of the natural bone formation process, a transient phase which is a precursor to hydroxyapatite (HA)[30–32].

These ACPs have been used in bone

cements to increase the cement reactivity or bioresorbability. ACPs have also been shown to increase osteoconductivity of implant coatings as well as increase the mechanical properties of composites[30]. Magnesium has been widely shown to stabilize calcium phosphate in the ACP stage over conversion to HA[33,34]. However, the analogy between ACP and amorphous magnesium phosphate is not clear as the synthesis of amorphous magnesium phosphate has only recently been reported[35]. In a recent study from our laboratory, a comparison of the cytocompatibility of dense substrates of amorphous magnesium phosphate and crystalline tri-magnesium phosphate showed increased proliferation and differentiation of murine 3

ACCEPTED MANUSCRIPT

pre-osteoblast cells on the amorphous substrates over the crystalline substrates[36]. It is however unclear whether the use of amorphous tri-magnesium phosphates is feasible to generate a functional bone cement product, or more importantly, how this cement may compare to cements made with a crystalline tri-magnesium phosphate. In this study, the functionality of these amorphous and crystalline tri-magnesium phosphate powders, as well as semi-crystalline tri-magnesium phosphates powders, in cement forming reactions with a concentrated, neutral

RI PT

pH ammonium phosphate solution for bone cement applications is explored and systematically assessed.

SC

2. Experimental 2.1. Magnesium phosphate powder synthesis

M AN U

Magnesium phosphate powders were synthesized via an aqueous precipitation reaction between 0.3 mol/L MgCl2 and 0.2 mol/L Na3PO4, in deionized water. The magnesium containing solution was pipetted into the phosphate containing solution, resulting in the precipitation of tri-magnesium phosphate (TMP), Mg3(PO4)2. The pH of the reacting solution was observed to be basic and was not controlled during the synthesis. The solution was centrifuged at 10,000 r/min for 5 min and the excess liquid was discarded. The TMP precipitate

TE D

was collected and rinsed thrice in deionized water followed by washing in acetone and finally dried overnight at 60 °C. Powders were then thermally treated at 400, 600 or 800 °C for 6 h at each temperature in a box furnace in air to form amorphous, semi-crystalline or fully

EP

crystalline dehydrated TMP, respectively. Powders were lightly ground and sieved to –500 µm to remove any large agglomerated clusters.

AC C

2.2. Magnesium phosphate powder characterization Magnesium phosphate as-synthesized and thermally treated powder samples were characterized utilizing a number of techniques. Thermogravimetric analysis and differential scanning calorimetry (TGA and DSC) were used to identify the level of hydration and temperature of crystallization utilizing a Netzsch STA 409 PC Luxx system. The as-synthesized powder was heated at a rate of 10 °C/min to 1000 °C under argon. Crystalline phases were identified by X-ray diffraction (XRD) using a Philips X-Pret PRO diffractometer equipped with CuKα radiation and X’celerator Si-detector. The X-ray generator was operated at 45 kV and 40 mA at a 2θ range of 10°–90°.

Fourier transform infrared spectroscopy (FTIR) was completed

on a Nicolet 6700 spectrophotometer with a diamond ATR Smart orbit. FTIR spectroscopy was 4

ACCEPTED MANUSCRIPT

used to identify the differences in molecular chemical linkages between amorphous and crystalline precipitates. Spectra were collected at 1.0 cm–1 resolution averaging 64 scans in the 400–4000 cm–1 frequency range.

Specific surface area (SSA) measurements was determined

from the N2 adsorption-desorption isotherms obtained using the Brunauer–Emmett–Teller (BET) method performed on a Micromeritics ASAP 2020 instrument. SSA measurements were used to determine the area, and subsequent relative reactivity of synthesized powders. Particles

RI PT

were observed by scanning electron microscopy (SEM) using a JEOL, JSM-6610LV system. Samples were sputter coated with palladium on a Cressington sputter coater 108A prior to imaging.

SC

2.3. Cement formation

Series of cements were compared by combining the amorphous, semi-crystalline and

M AN U

crystalline Mg3(PO4)2 powders with a pH 7.0, 3 mol/L solution of ammonium phosphate. The solution was made by combining monobasic ammonium phosphate (NH4H2PO4) and dibasic ammonium phosphate ((NH4)2HPO4) salts in deionized water at a ratio to obtain pH 7.0.

The

ratio of monobasic to dibasic ammonium phosphate to yield neutral pH 3 mol/L solution is approximately 2:7 monobasic:dibasic, which would yield X and Y of approximately 16 and 11,

TE D

respectively. Assuming an ideal complete reaction, this would yield a final product of approximately, 60% MgNH4PO4 (Struvite) and 40% MgHPO4 (Newberyite), as described below. The powders were mixed with the ammonium phosphate solution at a variety of powder-to-liquid ratios (P:L), and thus the effect of the powder state on the cement reactivity

EP

was assessed. The cementing reaction should proceed per the reaction (1) shown below for

AC C

crystalline, semi-crystalline and amorphous Mg3(PO4)2: Mg3(PO4)2 + (NH4)x(H)yPO4 = x·MgNH4PO4 + y·MgHPO4

(1)

where x and y are based on the mole balance of the monobasic and dibasic ammonium phosphate salts in solution. In addition, with the given P:L ratios, a molar ratio of magnesium phosphate to ammonium phosphate greater than one is always maintained. This is because for the acid-base reacting cements such as these, an excess of magnesium phosphate ensures that the reaction proceeds further towards completion, which should yield greater strength and reduce the leaching of remaining soluble salts into the media. The P:L ranges tested in this study are shown in Table 1. The cement nomenclature used is dictated by the thermal treatment temperature of the powder, namely 400, 600 or 800 °C cement. Additional detail, such as the

5

ACCEPTED MANUSCRIPT

powder-to-liquid ratio is explicitly stated. 2.4. Cement characterization

Cements have been made with powders heat treated at 400, 600 and 800 °C after a soak time of 6 h at each temperature, using a variety of powder-to-liquid ratios, as indicated in Table

RI PT

1. The setting time of the cement pastes were determined with the Gilmore needle test following the ASTM standard C266-13 at room temperature. Amorphous, semi-crystalline and crystalline tri-magnesium phosphate powders were mixed with the specified volume of solution and stirred for 60 s before filling into 10 mm circular molds. The handling characteristics were

SC

qualitatively assessed based on consistency and injectability. The set cements were stored overnight at 37 °C before subsequent testing. The compressive strength of cement cylinders,

M AN U

nominally 6 mm × 12 mm, was analyzed using a 2 kN load cell (Instron, Norwood, MA) and a cross head speed of 1.0 mm/min, with 3 samples per condition.

XRD and SEM analysis were

used to assess the cements following completion of the reaction, utilizing the same procedures and equipment specified above. Pellets of the reacted cement were soaked in phosphate buffered saline (PBS) for 48 h at 37 °C. At this point, the pH and qualitative strength of each formulation was recorded. XRD and SEM analyses were again conducted on the cements

3. Results and Discussion

TE D

following PBS incubation to determine if any changes occurred due to the saline incubation.

EP

3.1. Magnesium phosphate powder characterization Thermogravimetric analysis and the differential scanning calorimetry results of the

AC C

as-synthesized TMP powder can be seen in Fig. 1, showing the vast majority of weight loss occurring at temperatures lower than 200 °C, shown in TGA. The exothermic peak observed at temperatures less than 700 °C in the DSC scan, without any noticeable corresponding weight change, is attributed to the bulk crystallization of the material. These results confirmed the selection of appropriate temperatures of 400, 600 and 800 °C used for obtaining the dehydrated amorphous, semi-crystalline and fully crystalline TMP for subsequent cementing reactions. XRD patterns of the synthesized and heat treated powders, shown in Fig. 2, indicate that the synthesized powder is amorphous when generated, and retains the amorphous structure following thermal treatment at 400 °C. At 600 °C however, crystalline peaks appear but a definitive amorphous hump is still retained, and finally at 800 °C, as expected following the

6

ACCEPTED MANUSCRIPT

thermal analysis results, complete conversion to fully crystalline Mg3(PO4)2 (ICCD Card 00-033-0876) is observed. FTIR spectra, seen in Fig. 3, corroborate the TGA/DSC and XRD results. FTIR spectra show increased crystallinity with thermal treatment indicated by the more defined peak separations, from the as-synthesized material through the 800 °C thermal treatment, in the 1200 to 900 cm–1 and 700 to 450 cm–1 ranges which correspond to PO43– adsorption bands[36].

RI PT

Additionally, the 600 and 800 °C thermally treated powders show an absence of a broad hump in the 3500 to 2700 cm–1 range and the band at 1650 cm–1 which are indicative of adsorbed water and OH stretching bonds, respectively[36].

Specific surface area measurements, shown in Fig. 4, of the powders indicate that the

SC

as-synthesized material displays a high surface area of approximately 170 m2/g, with subsequent thermal treatment as expected, significantly reducing the surface area. The initial

M AN U

surface area indicates that the particles are likely in the nanometer size range. However, the SEM images shown in Fig. 5, demonstrate that the powders comprise much larger particles. Previous work from our laboratory indicate that these particles are in fact agglomerates of much smaller, nanometer-sized particles, resulting in the large macroscopic particulate clusters contributing to the measured surface area values.

TE D

3.2. Cement characterization

A variety of P:L ratios for each heat treated powder was tested, with the goal of achieving a viscous paste consistency with set time of 10 and 15 min. Table 1 contains the P:L ratios and

EP

the cement setting properties of the 400, 600, and 800 °C heat treated powders. The 400 and 600 °C powders were perceived to be highly reactive, requiring large amounts of solution to

AC C

wet the powder and create a paste. There was no P:L ratio tested for 400 and 600 °C powder which was within the ideal setting time parameters for a cement, namely forming a moldable paste with setting time of 10 and 15 min, as demonstrated in Fig. 6. The 800 °C heat treated powder created a more viable cement, with P:L of 1:1 creating a low viscosity paste with the most ideal initial (8ʹ53ʺ) and final (14ʹ47ʺ) set time. The high reactivity of the amorphous and semi-crystalline powders and the perceived increase from very short to long set time in these cements with increasing liquid content, is likely due to the complex interplay of the physicochemical properties of the powders and the resulting

cements.

TMP

powders

mixed

with

the

reacting

solution

undergo

a

dissolution-precipitation type reaction to generate the cemented product, wherein the TMP is

7

ACCEPTED MANUSCRIPT

partially dissolved into the solution, and then re-precipitates the product phase(s). Hurle et al. have recently shown that, for calcium phosphate cements, the amount of amorphous calcium phosphate plays a strong role in increasing the reactivity of the cement[37]. The authors deduced that the high reactivity of the amorphous calcium phosphate is due to the high, instantaneous dissolution of the amorphous phase into solution, resulting in supersaturated concentrations of ions in solution and earlier precipitation of the calcium-deficient hydroxyapatite. Additionally,

with

amorphous

calcium

phosphate,

due

to

the

high

RI PT

the authors determined that the cement crystallite size was reduced when cements were made super-saturation

conditions.

Super-saturation increases the nucleation probability and number of crystal nucleation sites, ultimately resulting in more, smaller crystals[37].

For the amorphous and semi-crystalline

SC

TMP cements, similar conditions and reactivity can be expected, with the amorphous TMP content quickly reacting and dissolving, causing super-saturation of the dissolved ion in the

M AN U

reacting solution, leading to rapid precipitation of the crystals, giving the cements the setting time less than 15 s (Fig. 6). With higher liquid volumes as expected, the second setting time increases to greater than 1 h while the first set time remains less than 2 min. This is likely due to the sheer volume of excess phosphate liquid used that cannot be completely consumed in the ensuing cement reaction since the formation of the crystals and the crystal growth alone

TE D

combined with the exothermic nature of the reaction cannot alone lead to complete solid phase formation. Evaporation of the excess cementing liquid is required to bring the crystals into contact and giving the cement mechanical resilience. The pH values of the cements generated with 400 and 600 °C heat treated TMP were

EP

lower than the cements from 800 °C TMP, also seen in Table 1. Additionally, the mechanical resilience of the cements from 400 and 600 °C heat treated TMP in PBS is quite low, as

AC C

demonstrated in Fig. 7. These cements were soft or easily friable after incubation in the saline solution, showing mechanical strengths of less than 5 MPa for all P:L, with strengths less than 2 MPa following PBS incubation. All cement samples demonstrated a loss of mechanical resilience with the PBS exposure. Opposed to this, the 800 °C TMP cements demonstrated much higher strengths, above 20 MPa, with P:L of at least 1:1. These cements showed marginal decrease in strength following PBS incubation, but remained significantly stronger than the native, as-reacted 400 and 600 °C TMP cements. Figs. 8 and 9 show the XRD results of the selected 400, 600, and 800 °C heat treated TMP cements, P:L of 1:2, 1:2 and 1:1, respectively, as generated (Fig. 8) and after PBS soaking (Fig. 9). The diffraction patterns of each cement after the final cement setting time show the formation of, instead of the predicted MgNH4PO4 (Struvite) and MgHPO4 (Newberyite), an 8

ACCEPTED MANUSCRIPT

intermediate phase, Mg3(NH4)2H4(PO4)4·8H2O (hannayite, ICCD 98-000-6099). The intensity of the peaks differs slightly between the starting TMP powders. In addition to this, the 800 °C precursor shows remaining unreacted Mg3(PO4)2 and the 400 °C and, to a lesser extent 600 °C heat treated TMP, show an underlying amorphous hump indicating remaining amorphous material, likely the unreacted amorphous TMP. Following incubation in PBS, all the cements transformed into the anticipated MgNH4PO4· 6H2O (ICCD 98-002-2086) and MgHPO4·3H2O

RI PT

(ICCD 00-035-0780), without any visible remaining crystalline or amorphous TMP, as shown in Fig. 9. This likely indicates that the cements, after setting, are not fully reacted, and the exposure to PBS allows for further reaction of the unreacted amorphous TMP as well as the transformation of the less stable hannayite phase to form the desired MgNH4PO4 phase.

SC

Figs. 10 and 11 indicate the FTIR results of the selected 400, 600, and 800 °C heat treated TMP precursor cements, P:L of 1:2, 1:2 and 1:1, respectively, as generated (Fig. 10) and after

M AN U

PBS soaking (Fig. 11). Similar to the powder FTIR spectra, cement FTIR spectra also show peaks surrounding the PO4 vibration frequencies (1200 to 900 cm–1 and 700 to 450 cm–1) and OH/HOH vibration regions, as well as NH4 vibration bands. As seen in previous studies, the poorly defined bands under the broad adsorbed water hump correspond to OH stretching vibrations (3250 cm–1) and NH4 stretching vibration (2900 cm–1)[36,38]. The bands between 1600 cm–1 and 1400 cm–1 are also contributed to NH4 vibrations, while the additional bands 650–750

TE D

cm–1 are attributed to water H bonding[38].

Unlike the XRD, the FTIR analysis shows no

significant change in the cements after incubation in PBS. This is likely due to the fact that FTIR assesses vibrational frequencies corresponding to the chemical moieties, and the P–O,

EP

H–O, and N–H bonds’ vibrational frequencies present in the structure are not changing significantly within the crystallographic and molecular structure.

AC C

In Fig. 12, SEM images of the cements after setting and following PBS incubation can be seen. Both native (external) surface and fracture surface images are shown. It can be seen that the internal and external surfaces of the 400 and 600 °C heat treated TMP precursor cements are quite different after setting. The native, external surfaces of these cements appear featureless, with almost wave-like topology containing embedded crystallites at 500× magnification particularly, for the 400 °C derived TMP cement, while the internal fracture surface displays more block shaped cuboidal shaped crystals. It is likely that this featureless surface of the cements is contributed by the amorphous content with precipitated extremely small crystallites embedded, as previously discussed, with no presence of crystalline facets emerging due to the rapid nature of the reaction. The fracture surface of the 400 and 600 °C

9

ACCEPTED MANUSCRIPT

TMP cement are similar to the 800 °C TMP cement, although the crystalline TMP appears to have resulted in a wider distribution of crystallite sizes in the cement structure. Unlike the amorphous and semi-crystalline TMP cements, the crystalline TMP results in cement with an external surface which is very similar to the internal fracture surface, showing block and elongated crystal growth, indicating a slower dissolution-precipitation reaction and more controlled crystal growth, as supported by the likely observed reaction rates. Following the

RI PT

incubation in PBS, the external morphology of the 400 and 600 °C heat treated TMP cements changes drastically, with the formation of a smaller cuboidal to spherical particles. These same shaped particles are seen in the fracture surface images as well. The 800 °C TMP cement appears to have undergone some formation of additional smaller particles at the expense of

SC

larger crystallites similar to Ostwald’s ripening, but the change is however, not as drastic. The significant changes seen in the 400 and 600 °C heat treated TMP cements likely play a role in

M AN U

the observed changes in mechanical strength as well as the changes in XRD. In all of these tests, it is not possible to separate the effect of powder crystallinity and the effects of powder surface area on the cement viability. However, in this study an attempt is made to correlate the physicochemical properties of the powders with the ensuing cementing reaction reactivity and cement functionality. XRD results show similar final product formation

TE D

for all the cement products while SEM and SSA results of reacted cements indicate differences in the cement crystallite size with the different starting powders. Thus far, the mechanical stability of the cements appears to depend on the rate of initial cementing reaction and cement bond formation, which in turn, is controlled by the physicochemical properties of the

EP

tri-magnesium phosphate powder. Although there are advantages and desirable properties achievable in amorphous precursor materials, viable cement could not be created within the

AC C

tested parameters of this study. Recently Bhaduri et al. utilized microwave-assisted aqueous synthesis of amorphous tri-magnesium phosphate, as well as microwave synthesis of magnesium hydroxide cements to alter the exothermicity of cements[12,39]. These synthesis techniques may be a potential solution to combat the high reactivity of amorphous tri-magnesium phosphate powders. Additional methods to reduce the reactivity of the amorphous TMP in cementing reactions include reduction in specific surface area through thermal treatment as well as the formation of densified agglomerates, through pressing of dense pellets and subsequent pulverization of these pellets, as well as the utilization of reaction retardants. Although it is not reported here, the first and third alternatives have been recently attempted in our laboratory although yielding similar results with no significant improvements in the mechanical resilience of the cements. The densification and pulverization method as well 10

ACCEPTED MANUSCRIPT

as a microwave synthesis method are still to be explored and will be reported in the near future. However, moving forward from the results presented here, future studies under these parameters will focus on the fully crystalline, low surface area tri-magnesium phosphate cement due to the simplicity and the observed overall efficiency of the approach.

RI PT

4. Conclusion Crystalline, semi-crystalline and amorphous tri-magnesium phosphate powders were tested with a neutral 3 mol/L ammonium phosphate solution in order to assess the cementing reactions of the various powders. Under the available parameters, the fully crystalline, low

SC

surface area 800 °C derived tri-magnesium phosphate powder resulted in cements which demonstrated better workability, more neutral pH and higher strength than the amorphous or semi-crystalline TMP derived cements. Future studies will focus on optimizing the cementing

will be reported subsequently. Acknowledgements

M AN U

reaction, microstructure, and studying the cytocompatibility of this cement formulation which

The authors gratefully acknowledge the financial support of the National Science

TE D

Foundation (Grant #0933153), Edward R. Weidlein Chair Professorship Funds, University of Pittsburgh, the Center for Complex Engineered Multifunctional Materials (CCEMM), University of Pittsburgh, and the National Science Foundation Graduate Research Fellowship Program (DGE-1247842). The authors also gratefully acknowledge the laboratory of Dr.

References

EP

Alejandro Almarza for use of mechanical testing equipment.

AC C

[1] T.W. Bauer, G.F. Muschler, Clin. Orthop. Relat. Res. 371 (2000) 10–27. [2] A. Kolk, J. Handschel, W. Drescher, D. Rothamel, F. Kloss, M. Blessmann, M. Heiland, K.D. Wolff, R. Smeets, J. Cranio-Maxill. Surg. 40 (2012) 706–718. [3] M.P. Hofmann, A.R. Mohammed, Y. Perrie, U. Gbureck, J.E. Barralet, Acta Biomater. 5 (2009) 43–49. [4] K.L. Low, S.H. Tan, S.H. Zein, J.A. Roether, V. Mourino, A.R. Boccaccini, J. Biomed. Mater. Res.-Part B 94 (2010) 273–286. [5] M.P. Ginebra, C. Canal, M. Espanol, D. Pastorino, E.B. Montufar, Adv. Drug Delivery Rev. 64 (2012) 1090–1110. [6] S.V. Dorozhkin, Biomaterials 31 (2010) 1465–1485.

11

ACCEPTED MANUSCRIPT

[7] M. Vallet-Regi, J.M. Gonzalez-Calbet, Prog. Solid State Chem. 32 (2004) 1–31. [8] G. Mestres, M. P. Ginebra, Acta Biomater. 7 (2011) 1853–1861. [9] G. Mestres, F.S. Aguilera, N. Manzanares, S. Sauro, R. Osorio, M. Toledano, M.P. Ginebra, Int. Endod. J. 47 (2014) 127–139. [10] G. Mestres, M. Abdolhosseini, W. Bowles, S.H. Huang, C. Aparicio, S.U. Gorr, M.P. Ginebra, Acta Biomater 9 (2013) 8384–8393.

RI PT

[11] M.P. Ginebra, M. Espanol, E.B. Montufar, R.A. Perez, G. Mestres, Acta Biomater. 6 (2010) 2863–2873.

[12] H. Zhou, A.K. Agarwal, V.K. Goel, S.B. Bhaduri, Mater. Sci. Eng. C 33 (2013) 4288–4294. [13] Y.L. Yu, J. Wang, C.S. Liu, B.W. Zhang, H.H. Chen, H. Guo, G.R. Zhong, W.D. Qu, S.H.

SC

Jiang, H.Y. Huang, Colloids Surf. B 76 (2010) 496–504.

[14] C. Moseke, V. Saratsis, U. Gbureck, J. Mater. Sci.-Mater. Med. 22 (2011) 2591–2598.

M AN U

[15] U. Klammert, A. Ignatius, U. Wolfram, T. Reuther, U. Gbureck, Acta Biomater. 7 (2011) 3469–3475.

[16] F.C.M. Driessens, M.G. Boltong, M.I. Zapatero, R.M.H. Verbeeck, W. Bonfield, O. Bermudez, E. Fernandez, M.P. Ginebra, J.A. Planell, J. Mater. Sci.-Mater. Med. 6 (1995) 272–278.

TE D

[17] W. Jahnen-Dechent, M. Ketteler, Clin. Kidney J. 5 (2012) 3–14. [18] W. Paul, C.P. Sharma, J. Mater. Sci.-Mater. Med. 18 (2007) 699–703. [19] S. Pina, J.M.F. Ferreira, Materials 3 (2010) 519–535. [20] S. Yoshizawa, A. Brown, A. Barchowsky, C. Sfeir, Connect. Tissue Res. 55 (2014)

EP

155–159.

[21] S. Yoshizawa, A. Brown, A. Barchowsky, C. Sfeir, Acta Biomater. 10 (2014) 2834–2842.

AC C

[22] F. Feyerabend, J. Fischer, J. Holtz, F. Witte, R. Willumeit, H. Druecker, C. Vogt, N. Hort, Acta Biomater. 6 (2010) 1834–1842. [23] N.J. Hallab, C. Vermes, C. Messina, K.A. Roebuck, T.T. Glant, J.J. Jacobs, J. Biomed. Mater. Res. 60 (2002) 420–433. [24] Y. Yu, J. Wang, C. Liu, B. Zhang, H. Chen, H. Guo, G. Zhong, W. Qu, S. Jiang, H. Huang, Colloids Surf. B 76 (2010) 496–504. [25] Ewald, K. Helmschrott, G. Knebl, N. Mehrban, L.M. Grover, U. Gbureck, J. Biomed. Mater. Res.-Part B. 96 (2011) 326–332. [26] U. Klammert, T. Reuther, M. Blank, I. Reske, J.E. Barralet, L.M. Grover, A.C. Kubler, U. Gbureck, Acta Biomater. 6 (2010) 1529–1535. [27] U. Klammert, E. Vorndran, T. Reuther, F.A. Mueller, K. Zorn, U. Gbureck, J. Mater. 12

ACCEPTED MANUSCRIPT

Sci.-Mater. Med. 21 (2010) 2947–2953.

[28] C. Grossardt, A. Ewald, L.M. Grover, J.E. Barralet, U. Gbureck, Tissue Eng. Part A 16 (2010) 3687–3695. [29] J.P. Schmitz, J.O. Hollinger, S.B. Milam, J. Oral Max. Surg. 57 (1999) 1122–1126. [30] C. Combes, C. Rey, Acta Biomater. 6 (2010) 3362–3378.

[32] S. Posner, F. Betts, Acc. Chem. Res. 8 (1975) 273–281.

RI PT

[31] S.V. Dorozhkin, Acta Biomater. 6 (2010) 4457–4475.

[33] N.C. Blumenthal, F. Betts, A.S. Posner, Calcif. Tissue Int. 23 (1977) 245–250. [34] D. Lee, P.N. Kumta, Mater. Sci. Eng. C 30 (2010) 1313–1317.

[35] H. Zhou, T.J.F. Luchini, S.B. Bhaduri, J. Mater. Sci.-Mater. Med. 23 (2012) 2831–2837.

SC

[36] L. Berzina-Cimdina, N. Borodajenko, in: T. Theophile (Ed), Infrared Spectroscopy Materials Science, Engineering and Technology, InTech, Online, 2012, pp. 123–148.

M AN U

[37] K. Hurle, J. Neubauer, M. Bohner, N. Doebelin, F. Goetz-Neunhoeffer, Acta Biomater. 10 (2014) 3931–3941.

[38] Zecchina, L. Marchese, S. Bordiga, C. Pazè, E. Gianotti, J. Phys. Chem. B 101 (1997) 10128–10135.

[39] H. Zhou, A.K. Agarwal, V.K. Goel, S.B. Bhaduri, Mater. Sci. Eng. C 33 (2013) 4288–4294.

TE D

[40] H. Zhou, S. Bhaduri, J. Biomed. Mater. Res.-Part B 100 (2012) 1142–1150.

Figure and table captions

EP

Table 1 Cement handling properties and pH in saline solution

Fig. 1 TGA and DSC curves corresponding to the synthesized Mg3(PO4)2 powders.

AC C

Fig. 2 XRD patterns of the synthesized Mg3(PO4)2 powders. Fig. 3 FTIR spectra corresponding to the various synthesized Mg3(PO4)2 powders. Fig. 4 BET measured surface area of the synthesized Mg3(PO4)2 powders. Fig. 5 SEM images of the synthesized and thermally treated Mg3(PO4)2 powders. Fig. 6 Setting time of the cements generated. Fig. 7 Uniaxial compression strengths corresponding to the various generated cements. Fig. 8 XRD patterns of 400, 600, and 800 °C cements, P:L of 1:2, 1:2 and 1:1, respectively after reaction completion. Fig. 9 XRD patterns of 400, 600, and 800 °C cements, P:L of 1:2, 1:2 and 1:1, respectively following 48 h PBS incubation.

13

ACCEPTED MANUSCRIPT Fig. 10 FTIR spectra of 400, 600, and 800 °C cements, P:L of 1:2, 1:2 and 1:1, respectively after reaction completion. Fig. 11 FTIR spectra of 400, 600, and 800 °C cements, P:L of 1:2, 1:2 and 1:1, respectively following 48 h PBS incubation. Fig. 12 SEM images of 400, 600, and 800 °C cements, P:L of 1:2, 1:2 and 1:1, respectively.

RI PT

The top group of images shows the fracture surface of cements, after reaction completion and after PBS incubation, while the bottom group of images shows the native (outside) surface of

Table list:

1:1 400 °C

1:2 2:5 1:3 1:1 1:2

600 °C 2:5

AC C

1:3 1:1 2:1

800 °C

Handling Reacts too quickly, cannot be stirred and molded to form a cement pellet Reacts very quickly, forms a dry, crumbled mixture which could be molded, but does not form a paste. Reacts quickly, but contains excess liquid to form a chunky paste that does not set. Reacts more slowly, forms a liquid paste that does not set. Reacts too quickly, cannot be stirred and molded to form a cement pellet Reacts very quickly, forms a dry, crumbled mixture which could be molded, but does not form a paste. Reacts very quickly, forms a dry, crumbled mixture which could be molded, but does not form a paste. Reacts quickly, but contains excess liquid to form a paste with large aggregates which does not set. Forms a nice putty, with desirable set times. Mixture is dry, forms a crumbled material, but packable into a mold and sets too quickly. Mixture is dry, forms a dry putty, packable into a mold and sets too quickly. Forms a very thin putty, set time is too long.

TE D

P:L ratio

EP

Powder

M AN U

Table 1

3:2 2:3

SC

the same cements, after reaction completion and after PBS incubation. (Scale bar: 10 µm).

14

pH in PBS N/A 6.02 5.99 5.96 6.87 6.35 6.27 6.15 7.47 7.58 7.77 6.57

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

Figure list:

AC C

EP

TE D

Fig. 1

Fig. 2

15

TE D

SC

M AN U

Fig. 3

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Fig. 4

Fig. 5

16

M AN U

SC

Fig. 6

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 7

Fig. 8

17

TE D

M AN U

Fig. 9

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Fig. 10

Fig. 11

18

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Fig. 12

19