- Email: [email protected]
A new way of forming a calcium phosphate cement using bioactive glasses as a reactive precursor
Author’s Accepted Manuscript A New Way Of Forming A Calcium Phosphate Cement Using Bioactive Glasses As A Reactive Precursor Niall W Kent, Robert G Hill, Natalia Karpukhina www.elsevier.com
PII: DOI: Reference:
S0167-577X(15)30610-8 http://dx.doi.org/10.1016/j.matlet.2015.09.099 MLBLUE19615
To appear in: Materials Letters Received date: 27 April 2015 Revised date: 10 August 2015 Accepted date: 25 September 2015 Cite this article as: Niall W Kent, Robert G Hill and Natalia Karpukhina, A New Way Of Forming A Calcium Phosphate Cement Using Bioactive Glasses As A Reactive Precursor, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2015.09.099 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 galley proof before it is published in its final citable 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.
A New Way Of Forming A Calcium Phosphate Cement Using Bioactive Glasses as a Reactive Precursor
Niall W Kent1, Robert G Hill and Natalia Karpukhina*
Dental Physical Sciences, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
*Corresponding author’s email: [email protected] Abstract Calcium phosphate cements (CPC)s are conventionally made by mixing crystalline calcium phosphates with aqueous solutions. In this study new CPCs are made by reacting bioactive glasses (BG)s with Ca(H2PO4)2 to form cement. It is found that high P2O5 content of 4 mol% or greater is required in BG to produce a cement. The phases formed are dependent on glass composition; brushite and octacalcium phosphate (OCP) form first with 6 mol% P2O5 BG. Brushite dissolves, reforms as OCP, then transforms to apatite. These new cements offer new route to forming CPC that combine in-situ setting and injectability of CPCs with resorbability and bioactivity of BGs.
Keywords:
1
The present address of this author is Department of Chemical Engineering, University College London, Torrington Place, London, United Kingdom
1
Calcium phosphate cement, apatite cement, brushite cement, bioactive glass 1 Introduction CPCs were proposed by Le Geros [1] and successfully developed by Brown and Chow [2], though the first cement formation in calcium phosphates system was demonstrated in 1950 by Kingery [3]. These cements are conventionally made by mixing crystalline calcium phosphate salts with an aqueous solution. The salts dissolve and re-precipitate as calcium deficient apatite (Ca10x(HPO4)x(PO4)6-x(OH)2-x･nH2O)
or Brushite also named as dicalcium phosphate
dihydrate (DCPD, CaHPO4.2H2O) [4]. Recently monetite cements have been also considered as an alternative to the brushite cements [5]. CPCs have found widespread use in orthopaedic surgery and cranial facial surgery as a bone substitute. Existing CPCs suffer from a number of disadvantages [6]; they are: i)
not very resorbable in-vivo, this is specifically related to the existing apatite cements,
ii)
prone to wash out on exposure to water before being completely set,
iii)
relatively brittle and have low compressive strengths.
In contrast bioactive glasses [7] have osteo-stimulatory properties and are more resorbable but generally cannot be injected through a narrow bore syringe and do not set hard in-vivo. Recently a number of studies have investigated the incorporation of BGs into existing CPC formulations [8-10], where the BG was of a large particle size and acted as a bioactive filler.
2
In this pilot study we investigated CPCs made from BGs where the glass was used as reactive precursor as opposed to filler and took part in the cement forming reaction. This approach has three major advantages: i)
The glass compositions are not restricted by the stoichiometry of crystal.
ii)
The dissolution rate/reactivity of BG can be controlled via glass composition/structure.
iii)
It is possible to incorporate a wide variety of therapeutic ions into the glass composition including: strontium to stimulate bone formation and provide radio-opacity [11], zinc for its beneficial effects for wound healing [12], cobalt to promote angiogenesis [13-14] and fluoride to up regulate osteoblasts and form fluorapatite [15-16].
In order to form cement from a BG it is important that the glass both dissolves and forms apatite rapidly. Recently it has been shown [17-19] that the speed of apatite formation with BGs is related to the glass network connectivity and the phosphate content. 2 Materials and Methods The glasses (Table 1) were melted at 1480 ˚C for one hour; with the synthesis details described earlier [11]. 100g of each glass was ground using a vibratory mill (Gy-Ro mill, Glen Creston) for 2x7 minutes and sieved to a particle size below 38 μm. The Ca(H2PO4)2 (Sigma-Aldrich) was prepared by grinding 27 g using the vibratory mill for 4 minutes.
3
The cement mixture was prepared by mixing the sieved glass powder with the milled Ca(H2PO4)2 powder with overall calcium to phosphorus ratio of 1.33, the stoichiometry of octacalcium phosphate (OCP, Ca8(HPO4)2(PO4)4･5H2O). The powders were hand mixed for 30s on a glass slab, with 2.5% Na2HPO4 solution with a liquid to powder ratio of 0.70ml/g. The setting time of each cement was measured using the Gilmore needle test according to the ISO 9917-1:2007(E). The compressive strength specimens were prepared according to the ISO 9917-1:2007(E) using split cylindrical moulds. The cements were stored at 37oC for two hours. Each cylinder was immersed in 10 ml of Tris buffer solution at 37oC for either of 1,24,168,672 hours prior to testing. Powder XRD was carried out for the cements after immersion using Bruker D8A25-Advance diffractometer with the CuKα radiation at 40kV and 40mA. The 31P MAS-NMR experiments were run on Bruker NMR spectrometer at the 242.9MHz frequency. The powder samples were packed into 4mm rotor and spun at 11-12kHz. The measurements were done using 60s recycle delay and 85% H3PO4 was used to reference the chemical shift scale. The fracture surface of the cement cylinders were gold coated and examined using Hitachi S-3400 machine with the accelerating voltage at 20kV and an emission current of 54mA. 3 Results Table 1 gives the initial and final setting times. The two cements P4 and P6 were produced as cylinders for the compressive strength experiments.
4
However, the P4 cements disintegrated upon immersion and only the compressive strength of P6 cements was determined as shown in Table 2. The XRD for the P4 and P6 cements are shown in Figure 1. At 1Hr Brushite is present for P4 (Figure 1a). On increasing the immersion time the Brushite dissolves and is progressively replaced by OCP. OCP has an almost identical diffraction pattern to hydroxyapatite but also exhibits a diffraction line at 4.68o 2 corresponding to the 100 water inter layer spacing (18.6 Å) [20-21]. The P6 cement also shows the presence of Brushite at 1Hr and 24Hrs whilst the 4.68o 2 line of OCP is present at 1,24 and 168Hrs but is absent at 672Hrs (Figure 1b). Figure 2 shows the 31P MAS-NMR results for P4 and P6 glasses and cements. The assignment of the spectra was done based on the previous studies [22-25]. The glasses exhibit a broad peak at ≈5 ppm corresponding to mixed sodium calcium amorphous orthophosphate. In Figure 2a the dominant peak at 1.4 ppm for the P4 at 1Hr and 24Hrs indicates that Brushite is the primary phase. A small fraction of the signal at -1.4 and -0.2 ppm indicates that Monetite (CaHPO4) is also present. At 168Hrs chemical shifts at -0.2,2.0,3.2 and 3.6 ppm are present indicative of the formation of OCP, in addition to Brushite and Monetite. At 672Hrs peaks at -0.2,2.0,3.2 and 3.6 ppm are found showing the presence of OCP. Figure 2b shows chemical shifts at -1.4,-0.2,1.4 and 3.3/3.1 ppm in 1Hr and 24Hr samples, these are Monetite (-1.4,-0.2 ppm), Brushite (1.4 ppm) and Apatite (3.1 ppm). The 672Hr sample has a chemical shift at 2.9 ppm assigned as apatite.
5
The scanning electron micrographs (SEM) of the P4 and P6 cements are shown in Figures 3 and 4. SEM showed porous structure with the crystals morphology and size changing over time. Small thin plate crystals were seen at 1Hr in P4 cements. This changes to extremely elongated ribbon- or blade-like crystals at 168 Hr with the structure becomes much more open and remains the same by 672Hr. In P6 cements with less open structure, a mixture of the crystals morphologies has been seen. The smaller whiskers-like crystals that were initially present disappeared with time and only plate-like crystals were remained.
4 Discussion The two glasses with the lowest phosphate contents were not capable of forming cements that set within 90 minutes. The two higher phosphate content glasses gave set cements and the initial and final set decreased with increasing phosphate content. This suggests that the high phosphate content of the BG, not less than 4% P2O5, enables entirely formation of CPCs via this newly proposed route. No signs of the Ca(H2PO4)2 or its hydrates have been seen from both XRD and NMR. NMR showed that the broad signal from the glass has disappeared in cements indicating all the phosphorus from the both sources has completely reacted before 1Hr . XRD and NMR revealed that the phases formed in the cements were dependent on the glass composition. Initial formation of brushite in both formulations is controlled by an acidity resulted from the dissolution of the
6
soluble calcium phosphate. However, early formation of OCP and transformation into apatite, seen in P6, is clearly related to the high phosphate content in the glass. Small thin plate crystals initially seen P4 cements have morphology that was reported earlier for brushite [26]. The dramatic change of the microstructure at 168 Hr due to formation of blade like shape crystals known for the OCP [27] prevail the cements to sustain any strength without optimisation. The microstructure of the P6 formulation sustain a mechanical load; the P6 cement exhibited a compressive strength higher than trabecular bone [28]. However, the drop of the strength observed after 168Hrs is thought to occur due to a volume reduction associated with formation of apatite.
5 Conclusions CPCs can be formed from using BGs with high P2O5 and soluble calcium hydrogen phosphate on mixing with water. This study demonstrates a new route to forming CPC using BGs as reactive precursors as an alternative to forming cements with mixtures of crystalline calcium phosphate salts.
Acknowledgements Prof Kunio Ishikawa (Kyushu University) is gratefully acknowledged for hosting Japanese Society for the Promotion of Science (JSPS) short-term fellowship.
7
The UK MRC, Barts and The London School of Medicine and Dentistry, JSPS are gratefully acknowledged for funding.
8
References
[1] Legeros RZ, Chohayeb A, Schulman A. Apatitic calcium phosphates possible dental restorative materials. Journal of Dental Research. 1982;61:343. [2] Brown WE, Chow LC. A New Calcium-Phosphate Setting Cement. Journal of Dental Research. 1983;62:672-. [3] Kingery WD. II, Cold-Setting Properties. Journal of the American Ceramic Society. 1950;33:242-6. [4] Driessens FCM, Boltong MG, Bermudez O, Planell JA, Ginebra MP, Fernandez E. Effective Formulations for the Preparation of Calcium-Phosphate Bone Cements. J Mater Sci-Mater M. 1994;5:164-70. [5] Cama G, Gharibi B, Sait MS, Knowles JC, Lagazzo A, Romeed S, et al. A novel method of forming micro- and macroporous monetite cements. J Mat Chem B. 2013;1:958-69. [6] Bohner M, Gbureck U, Barralet JE. Technological issues for the development of more efficient calcium phosphate bone cements: A critical assessment. Biomaterials. 2005;26:6423-9. [7] Jones JR. Review of bioactive glass: From Hench to hybrids. Acta Biomater. 2013;9:4457-86. [8] Renno ACM, van de Watering FCJ, Nejadnik MR, Crovace MC, Zanotto ED, Wolke JGC, et al. Incorporation of bioactive glass in calcium phosphate cement: An evaluation. Acta Biomater. 2013;9:5728-39. [9] Renno ACM, Nejadnik MR, van de Watering FCJ, Crovace MC, Zanotto ED, Hoefnagels JPM, et al. Incorporation of bioactive glass in calcium phosphate cement: Material characterization and in vitro degradation. J Biomed Mater Res A. 2013;101A:2365-73. [10] Yu L, Li Y, Zhao K, Tang YF, Cheng Z, Chen J, et al. A Novel Injectable Calcium Phosphate Cement-Bioactive Glass Composite for Bone Regeneration. Plos One. 2013;8. [11] Fujikura K, Karpukhina N, Kasuga T, Brauer DS, Hill RG, Law RV. Influence of strontium substitution on structure and crystallisation of Bioglass (R) 45S5. J Mater Chem. 2012;22:7395-402. [12] Agren MS. Studies on Zinc in Wound-Healing. Acta Derm-Venereol. 1990:1-36. [13] Azevedo MM, Jell G, O'Donnell MD, Law RV, Hill RG, Stevens MM. Synthesis and characterization of hypoxia-mimicking bioactive glasses for skeletal regeneration. J Mater Chem. 2010;20:8854-64. [14] Buttyan R, Chichester P, Stisser B, Matsumoto S, Ghafar MA, Levin RM. Acute intravesical infusion of a cobalt solution stimulates a hypoxia response, growth and angiogenesis in the rat bladder. J Urology. 2003;169:2402-6. [15] Gentleman E, Stevens MM, Hill RG, Brauer DS. Surface properties and ion release from fluoride-containing bioactive glasses promote osteoblast differentiation and mineralization in vitro. Acta Biomater. 2013;9:5771-9. [16] Chen XJ, Chen XH, Brauer DS, Wilson RM, Hill RG, Karpukhina N. Bioactivity of Sodium Free Fluoride Containing Glasses and Glass-Ceramics. Materials. 2014;7:5470-87.
9
[17] O'Donnell MD, Watts SJ, Law RV, Hill RG. Effect of P2O5 content in two series of soda lime phosphosilicate glasses on structure and properties - Part II: Physical properties. J Non-Cryst Solids. 2008;354:3561-6. [18] Eden M. The split network analysis for exploring composition-structure correlations in multi-component glasses: I. Rationalizing bioactivity-composition trends of bioglasses. J Non-Cryst Solids. 2011;357:1595-602. [19] Hill RG, Brauer DS. Predicting the bioactivity of glasses using the network connectivity or split network models. J Non-Cryst Solids. 2011;357:3884-7. [20] Mathew M, Brown WE, Schroeder LW, Dickens B. Crystal-structure of octacalcium bis(hydrogenphosphate) tetrakis(phosphate)pentahydrate, Ca8(HPO4)2(PO4)4.5H2O. Journal of Crystallographic and Spectroscopic Research. 1988;18:235-50. [21] Yokoi T, Kamitakahara M, Ohtsuki C. Continuous expansion of the interplanar spacing of octacalcium phosphate by incorporation of dicarboxylate ions with a side chain. Dalton Transactions. 2015. [22] Rothwell WP, Waugh JS, Yesinowski JP. High-resolution variabletemperature P-31 NMR of solid calcium phosphates. J Am Chem Soc. 1980;102:2637-43. [23] Tseng YH, Mou CY, Chan JCC. Solid-state NMR study of the transformation of octacalcium phosphate to hydroxyapatite: A mechanistic model for central dark line formation. J Am Chem Soc. 2006;128:6909-18. [24] Pourpoint F, Diogo CC, Gervais C, Bonhomme C, Fayon F, Dalicieux SL, et al. High-resolution solid state NMR experiments for the characterization of calcium phosphate biomaterials and biominerals. J Mater Res. 2011;26:235568. [25] Davies E, Duer MJ, Ashbrook SE, Griffin JM. Applications of NMR Crystallography to Problems in Biomineralization: Refinement of the Crystal Structure and P-31 Solid-State NMR Spectral Assignment of Octacalcium Phosphate. J Am Chem Soc. 2012;134:12508-15. [26] Kumar M, Xie J, Chittur K, Riley C. Transformation of modified brushite to hydroxyapatite in aqueous solution: effects of potassium substitution. Biomaterials. 1999;20:1389-99. [27] Legeros RZ. Ppreparation of octacalcium phosphate (OCP) - a direct fast method. Calcif Tissue Int. 1985;37:194-7. [28] Misch CE, Qu ZM, Bidez MW. Mechanical properties of trabecular bone in the human mandible: Implications for dental implant treatment planning and. Surgical placement. J Oral Maxillofac Surg. 1999;57:700-6.
Highlights
A new way of making calcium phosphate bone cements is proposed
10
New calcium phosphate cement is formed as a result of reaction between bioactive glass and soluble calcium phosphate in solution The properties of the cements can be controlled via the glass composition The phases desired to be in the final set cements can be regulated via the glass composition
11
Table 1 Glass compositions in mol% and initial and final setting time in minutes for the experimental cements. Theoretical network connectivity of all glass compositions is equal to 2.00.
Glass
SiO2
P2O5
CaO
Na2O Initial Set Final Set
P0
50.0
0.0
45.0
5.0
>90
>90
P2
46.0
2.0
46.8
5.2
>90
>90
P4
42.0
4.0
48.6
5.4
35.5
89.0
P6
38.0
6.0
50.4
5.6
10.5
15.0
Table 2 Compressive strength in MPa of the P6 cements after each of the immersion period in Tris buffer (Figures in brackets give the SD for n=8)
Glass/Time
1Hr
24Hrs
168Hrs
672Hrs
P6
9.65 (0.56) 12.90 (1.60) 10.30 (1.33) 5.67(0.94)
12
Figure captions
Figure 1. X-ray diffraction patterns of cement formulation produced from glass P4 (a) and P6 (b) after immersion in Tris buffer solution for (i) 1 hour, (ii) 24 hours, (iii) 168 hours and (iv) 672 hours. ▲ – Brushite or DCPD; ▼ – Monetite or DCPA; ● – OCP; + Apatite.
Figure 2. 31P MAS-NMR spectra of the starting glass powder and Tris immersed cement samples in the cement system produced from glasses P4 (a) and P6 (b) showing (i) initial glass powder; (ii) 1 hour; (iii) 24 hours; (iv) 168 hours; (v) 672 hours.
Figure 3 Scanning electron photomicrographs of P4 cements at 1 hour (top left), 24 hours (top right), 168 hours (bottom left) and 672 hours (bottom right).
Figure 4 Scanning electron photomicrographs of P6 cements at 1 hour (top left), 24 hours (top right), 168 hours (bottom left) and 672 hours (bottom right).
13
Figure 1(a)
Rel. Intensity
iv
iii
ii
i 0
5
10
15
20
25
30
35
40
2Theta, degrees
14
Figure 1(b)
+
+ iv
Rel. Intensity
++
+
+
+
iii
ii
i 0
5
10
15
20
25
30
35
40
2Theta, degrees
15
Figure 2(a)
v
iv iii ii i
31P
chemical shift, ppm
16
Figure 2(b)
v iv iii
ii
i 31P
chemical shift, ppm
17
Figure 3
18
Figure 4
19
PII: DOI: Reference:
S0167-577X(15)30610-8 http://dx.doi.org/10.1016/j.matlet.2015.09.099 MLBLUE19615
To appear in: Materials Letters Received date: 27 April 2015 Revised date: 10 August 2015 Accepted date: 25 September 2015 Cite this article as: Niall W Kent, Robert G Hill and Natalia Karpukhina, A New Way Of Forming A Calcium Phosphate Cement Using Bioactive Glasses As A Reactive Precursor, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2015.09.099 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 galley proof before it is published in its final citable 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.
A New Way Of Forming A Calcium Phosphate Cement Using Bioactive Glasses as a Reactive Precursor
Niall W Kent1, Robert G Hill and Natalia Karpukhina*
Dental Physical Sciences, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
*Corresponding author’s email: [email protected] Abstract Calcium phosphate cements (CPC)s are conventionally made by mixing crystalline calcium phosphates with aqueous solutions. In this study new CPCs are made by reacting bioactive glasses (BG)s with Ca(H2PO4)2 to form cement. It is found that high P2O5 content of 4 mol% or greater is required in BG to produce a cement. The phases formed are dependent on glass composition; brushite and octacalcium phosphate (OCP) form first with 6 mol% P2O5 BG. Brushite dissolves, reforms as OCP, then transforms to apatite. These new cements offer new route to forming CPC that combine in-situ setting and injectability of CPCs with resorbability and bioactivity of BGs.
Keywords:
1
The present address of this author is Department of Chemical Engineering, University College London, Torrington Place, London, United Kingdom
1
Calcium phosphate cement, apatite cement, brushite cement, bioactive glass 1 Introduction CPCs were proposed by Le Geros [1] and successfully developed by Brown and Chow [2], though the first cement formation in calcium phosphates system was demonstrated in 1950 by Kingery [3]. These cements are conventionally made by mixing crystalline calcium phosphate salts with an aqueous solution. The salts dissolve and re-precipitate as calcium deficient apatite (Ca10x(HPO4)x(PO4)6-x(OH)2-x･nH2O)
or Brushite also named as dicalcium phosphate
dihydrate (DCPD, CaHPO4.2H2O) [4]. Recently monetite cements have been also considered as an alternative to the brushite cements [5]. CPCs have found widespread use in orthopaedic surgery and cranial facial surgery as a bone substitute. Existing CPCs suffer from a number of disadvantages [6]; they are: i)
not very resorbable in-vivo, this is specifically related to the existing apatite cements,
ii)
prone to wash out on exposure to water before being completely set,
iii)
relatively brittle and have low compressive strengths.
In contrast bioactive glasses [7] have osteo-stimulatory properties and are more resorbable but generally cannot be injected through a narrow bore syringe and do not set hard in-vivo. Recently a number of studies have investigated the incorporation of BGs into existing CPC formulations [8-10], where the BG was of a large particle size and acted as a bioactive filler.
2
In this pilot study we investigated CPCs made from BGs where the glass was used as reactive precursor as opposed to filler and took part in the cement forming reaction. This approach has three major advantages: i)
The glass compositions are not restricted by the stoichiometry of crystal.
ii)
The dissolution rate/reactivity of BG can be controlled via glass composition/structure.
iii)
It is possible to incorporate a wide variety of therapeutic ions into the glass composition including: strontium to stimulate bone formation and provide radio-opacity [11], zinc for its beneficial effects for wound healing [12], cobalt to promote angiogenesis [13-14] and fluoride to up regulate osteoblasts and form fluorapatite [15-16].
In order to form cement from a BG it is important that the glass both dissolves and forms apatite rapidly. Recently it has been shown [17-19] that the speed of apatite formation with BGs is related to the glass network connectivity and the phosphate content. 2 Materials and Methods The glasses (Table 1) were melted at 1480 ˚C for one hour; with the synthesis details described earlier [11]. 100g of each glass was ground using a vibratory mill (Gy-Ro mill, Glen Creston) for 2x7 minutes and sieved to a particle size below 38 μm. The Ca(H2PO4)2 (Sigma-Aldrich) was prepared by grinding 27 g using the vibratory mill for 4 minutes.
3
The cement mixture was prepared by mixing the sieved glass powder with the milled Ca(H2PO4)2 powder with overall calcium to phosphorus ratio of 1.33, the stoichiometry of octacalcium phosphate (OCP, Ca8(HPO4)2(PO4)4･5H2O). The powders were hand mixed for 30s on a glass slab, with 2.5% Na2HPO4 solution with a liquid to powder ratio of 0.70ml/g. The setting time of each cement was measured using the Gilmore needle test according to the ISO 9917-1:2007(E). The compressive strength specimens were prepared according to the ISO 9917-1:2007(E) using split cylindrical moulds. The cements were stored at 37oC for two hours. Each cylinder was immersed in 10 ml of Tris buffer solution at 37oC for either of 1,24,168,672 hours prior to testing. Powder XRD was carried out for the cements after immersion using Bruker D8A25-Advance diffractometer with the CuKα radiation at 40kV and 40mA. The 31P MAS-NMR experiments were run on Bruker NMR spectrometer at the 242.9MHz frequency. The powder samples were packed into 4mm rotor and spun at 11-12kHz. The measurements were done using 60s recycle delay and 85% H3PO4 was used to reference the chemical shift scale. The fracture surface of the cement cylinders were gold coated and examined using Hitachi S-3400 machine with the accelerating voltage at 20kV and an emission current of 54mA. 3 Results Table 1 gives the initial and final setting times. The two cements P4 and P6 were produced as cylinders for the compressive strength experiments.
4
However, the P4 cements disintegrated upon immersion and only the compressive strength of P6 cements was determined as shown in Table 2. The XRD for the P4 and P6 cements are shown in Figure 1. At 1Hr Brushite is present for P4 (Figure 1a). On increasing the immersion time the Brushite dissolves and is progressively replaced by OCP. OCP has an almost identical diffraction pattern to hydroxyapatite but also exhibits a diffraction line at 4.68o 2 corresponding to the 100 water inter layer spacing (18.6 Å) [20-21]. The P6 cement also shows the presence of Brushite at 1Hr and 24Hrs whilst the 4.68o 2 line of OCP is present at 1,24 and 168Hrs but is absent at 672Hrs (Figure 1b). Figure 2 shows the 31P MAS-NMR results for P4 and P6 glasses and cements. The assignment of the spectra was done based on the previous studies [22-25]. The glasses exhibit a broad peak at ≈5 ppm corresponding to mixed sodium calcium amorphous orthophosphate. In Figure 2a the dominant peak at 1.4 ppm for the P4 at 1Hr and 24Hrs indicates that Brushite is the primary phase. A small fraction of the signal at -1.4 and -0.2 ppm indicates that Monetite (CaHPO4) is also present. At 168Hrs chemical shifts at -0.2,2.0,3.2 and 3.6 ppm are present indicative of the formation of OCP, in addition to Brushite and Monetite. At 672Hrs peaks at -0.2,2.0,3.2 and 3.6 ppm are found showing the presence of OCP. Figure 2b shows chemical shifts at -1.4,-0.2,1.4 and 3.3/3.1 ppm in 1Hr and 24Hr samples, these are Monetite (-1.4,-0.2 ppm), Brushite (1.4 ppm) and Apatite (3.1 ppm). The 672Hr sample has a chemical shift at 2.9 ppm assigned as apatite.
5
The scanning electron micrographs (SEM) of the P4 and P6 cements are shown in Figures 3 and 4. SEM showed porous structure with the crystals morphology and size changing over time. Small thin plate crystals were seen at 1Hr in P4 cements. This changes to extremely elongated ribbon- or blade-like crystals at 168 Hr with the structure becomes much more open and remains the same by 672Hr. In P6 cements with less open structure, a mixture of the crystals morphologies has been seen. The smaller whiskers-like crystals that were initially present disappeared with time and only plate-like crystals were remained.
4 Discussion The two glasses with the lowest phosphate contents were not capable of forming cements that set within 90 minutes. The two higher phosphate content glasses gave set cements and the initial and final set decreased with increasing phosphate content. This suggests that the high phosphate content of the BG, not less than 4% P2O5, enables entirely formation of CPCs via this newly proposed route. No signs of the Ca(H2PO4)2 or its hydrates have been seen from both XRD and NMR. NMR showed that the broad signal from the glass has disappeared in cements indicating all the phosphorus from the both sources has completely reacted before 1Hr . XRD and NMR revealed that the phases formed in the cements were dependent on the glass composition. Initial formation of brushite in both formulations is controlled by an acidity resulted from the dissolution of the
6
soluble calcium phosphate. However, early formation of OCP and transformation into apatite, seen in P6, is clearly related to the high phosphate content in the glass. Small thin plate crystals initially seen P4 cements have morphology that was reported earlier for brushite [26]. The dramatic change of the microstructure at 168 Hr due to formation of blade like shape crystals known for the OCP [27] prevail the cements to sustain any strength without optimisation. The microstructure of the P6 formulation sustain a mechanical load; the P6 cement exhibited a compressive strength higher than trabecular bone [28]. However, the drop of the strength observed after 168Hrs is thought to occur due to a volume reduction associated with formation of apatite.
5 Conclusions CPCs can be formed from using BGs with high P2O5 and soluble calcium hydrogen phosphate on mixing with water. This study demonstrates a new route to forming CPC using BGs as reactive precursors as an alternative to forming cements with mixtures of crystalline calcium phosphate salts.
Acknowledgements Prof Kunio Ishikawa (Kyushu University) is gratefully acknowledged for hosting Japanese Society for the Promotion of Science (JSPS) short-term fellowship.
7
The UK MRC, Barts and The London School of Medicine and Dentistry, JSPS are gratefully acknowledged for funding.
8
References
[1] Legeros RZ, Chohayeb A, Schulman A. Apatitic calcium phosphates possible dental restorative materials. Journal of Dental Research. 1982;61:343. [2] Brown WE, Chow LC. A New Calcium-Phosphate Setting Cement. Journal of Dental Research. 1983;62:672-. [3] Kingery WD. II, Cold-Setting Properties. Journal of the American Ceramic Society. 1950;33:242-6. [4] Driessens FCM, Boltong MG, Bermudez O, Planell JA, Ginebra MP, Fernandez E. Effective Formulations for the Preparation of Calcium-Phosphate Bone Cements. J Mater Sci-Mater M. 1994;5:164-70. [5] Cama G, Gharibi B, Sait MS, Knowles JC, Lagazzo A, Romeed S, et al. A novel method of forming micro- and macroporous monetite cements. J Mat Chem B. 2013;1:958-69. [6] Bohner M, Gbureck U, Barralet JE. Technological issues for the development of more efficient calcium phosphate bone cements: A critical assessment. Biomaterials. 2005;26:6423-9. [7] Jones JR. Review of bioactive glass: From Hench to hybrids. Acta Biomater. 2013;9:4457-86. [8] Renno ACM, van de Watering FCJ, Nejadnik MR, Crovace MC, Zanotto ED, Wolke JGC, et al. Incorporation of bioactive glass in calcium phosphate cement: An evaluation. Acta Biomater. 2013;9:5728-39. [9] Renno ACM, Nejadnik MR, van de Watering FCJ, Crovace MC, Zanotto ED, Hoefnagels JPM, et al. Incorporation of bioactive glass in calcium phosphate cement: Material characterization and in vitro degradation. J Biomed Mater Res A. 2013;101A:2365-73. [10] Yu L, Li Y, Zhao K, Tang YF, Cheng Z, Chen J, et al. A Novel Injectable Calcium Phosphate Cement-Bioactive Glass Composite for Bone Regeneration. Plos One. 2013;8. [11] Fujikura K, Karpukhina N, Kasuga T, Brauer DS, Hill RG, Law RV. Influence of strontium substitution on structure and crystallisation of Bioglass (R) 45S5. J Mater Chem. 2012;22:7395-402. [12] Agren MS. Studies on Zinc in Wound-Healing. Acta Derm-Venereol. 1990:1-36. [13] Azevedo MM, Jell G, O'Donnell MD, Law RV, Hill RG, Stevens MM. Synthesis and characterization of hypoxia-mimicking bioactive glasses for skeletal regeneration. J Mater Chem. 2010;20:8854-64. [14] Buttyan R, Chichester P, Stisser B, Matsumoto S, Ghafar MA, Levin RM. Acute intravesical infusion of a cobalt solution stimulates a hypoxia response, growth and angiogenesis in the rat bladder. J Urology. 2003;169:2402-6. [15] Gentleman E, Stevens MM, Hill RG, Brauer DS. Surface properties and ion release from fluoride-containing bioactive glasses promote osteoblast differentiation and mineralization in vitro. Acta Biomater. 2013;9:5771-9. [16] Chen XJ, Chen XH, Brauer DS, Wilson RM, Hill RG, Karpukhina N. Bioactivity of Sodium Free Fluoride Containing Glasses and Glass-Ceramics. Materials. 2014;7:5470-87.
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[17] O'Donnell MD, Watts SJ, Law RV, Hill RG. Effect of P2O5 content in two series of soda lime phosphosilicate glasses on structure and properties - Part II: Physical properties. J Non-Cryst Solids. 2008;354:3561-6. [18] Eden M. The split network analysis for exploring composition-structure correlations in multi-component glasses: I. Rationalizing bioactivity-composition trends of bioglasses. J Non-Cryst Solids. 2011;357:1595-602. [19] Hill RG, Brauer DS. Predicting the bioactivity of glasses using the network connectivity or split network models. J Non-Cryst Solids. 2011;357:3884-7. [20] Mathew M, Brown WE, Schroeder LW, Dickens B. Crystal-structure of octacalcium bis(hydrogenphosphate) tetrakis(phosphate)pentahydrate, Ca8(HPO4)2(PO4)4.5H2O. Journal of Crystallographic and Spectroscopic Research. 1988;18:235-50. [21] Yokoi T, Kamitakahara M, Ohtsuki C. Continuous expansion of the interplanar spacing of octacalcium phosphate by incorporation of dicarboxylate ions with a side chain. Dalton Transactions. 2015. [22] Rothwell WP, Waugh JS, Yesinowski JP. High-resolution variabletemperature P-31 NMR of solid calcium phosphates. J Am Chem Soc. 1980;102:2637-43. [23] Tseng YH, Mou CY, Chan JCC. Solid-state NMR study of the transformation of octacalcium phosphate to hydroxyapatite: A mechanistic model for central dark line formation. J Am Chem Soc. 2006;128:6909-18. [24] Pourpoint F, Diogo CC, Gervais C, Bonhomme C, Fayon F, Dalicieux SL, et al. High-resolution solid state NMR experiments for the characterization of calcium phosphate biomaterials and biominerals. J Mater Res. 2011;26:235568. [25] Davies E, Duer MJ, Ashbrook SE, Griffin JM. Applications of NMR Crystallography to Problems in Biomineralization: Refinement of the Crystal Structure and P-31 Solid-State NMR Spectral Assignment of Octacalcium Phosphate. J Am Chem Soc. 2012;134:12508-15. [26] Kumar M, Xie J, Chittur K, Riley C. Transformation of modified brushite to hydroxyapatite in aqueous solution: effects of potassium substitution. Biomaterials. 1999;20:1389-99. [27] Legeros RZ. Ppreparation of octacalcium phosphate (OCP) - a direct fast method. Calcif Tissue Int. 1985;37:194-7. [28] Misch CE, Qu ZM, Bidez MW. Mechanical properties of trabecular bone in the human mandible: Implications for dental implant treatment planning and. Surgical placement. J Oral Maxillofac Surg. 1999;57:700-6.
Highlights
A new way of making calcium phosphate bone cements is proposed
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New calcium phosphate cement is formed as a result of reaction between bioactive glass and soluble calcium phosphate in solution The properties of the cements can be controlled via the glass composition The phases desired to be in the final set cements can be regulated via the glass composition
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Table 1 Glass compositions in mol% and initial and final setting time in minutes for the experimental cements. Theoretical network connectivity of all glass compositions is equal to 2.00.
Glass
SiO2
P2O5
CaO
Na2O Initial Set Final Set
P0
50.0
0.0
45.0
5.0
>90
>90
P2
46.0
2.0
46.8
5.2
>90
>90
P4
42.0
4.0
48.6
5.4
35.5
89.0
P6
38.0
6.0
50.4
5.6
10.5
15.0
Table 2 Compressive strength in MPa of the P6 cements after each of the immersion period in Tris buffer (Figures in brackets give the SD for n=8)
Glass/Time
1Hr
24Hrs
168Hrs
672Hrs
P6
9.65 (0.56) 12.90 (1.60) 10.30 (1.33) 5.67(0.94)
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Figure captions
Figure 1. X-ray diffraction patterns of cement formulation produced from glass P4 (a) and P6 (b) after immersion in Tris buffer solution for (i) 1 hour, (ii) 24 hours, (iii) 168 hours and (iv) 672 hours. ▲ – Brushite or DCPD; ▼ – Monetite or DCPA; ● – OCP; + Apatite.
Figure 2. 31P MAS-NMR spectra of the starting glass powder and Tris immersed cement samples in the cement system produced from glasses P4 (a) and P6 (b) showing (i) initial glass powder; (ii) 1 hour; (iii) 24 hours; (iv) 168 hours; (v) 672 hours.
Figure 3 Scanning electron photomicrographs of P4 cements at 1 hour (top left), 24 hours (top right), 168 hours (bottom left) and 672 hours (bottom right).
Figure 4 Scanning electron photomicrographs of P6 cements at 1 hour (top left), 24 hours (top right), 168 hours (bottom left) and 672 hours (bottom right).
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Figure 1(a)
Rel. Intensity
iv
iii
ii
i 0
5
10
15
20
25
30
35
40
2Theta, degrees
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Figure 1(b)
+
+ iv
Rel. Intensity
++
+
+
+
iii
ii
i 0
5
10
15
20
25
30
35
40
2Theta, degrees
15
Figure 2(a)
v
iv iii ii i
31P
chemical shift, ppm
16
Figure 2(b)
v iv iii
ii
i 31P
chemical shift, ppm
17
Figure 3
18
Figure 4
19