Sr) Codoped Biphasic Calcium Phosphate with Tailored

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(Fe/Sr) co-doped biphasic calcium phosphate with tailored osteoblast cell functionality Subhadip Basu, Aritri Ghosh, Ananya Barui, and Bikramjit Basu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00813 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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ACS Biomaterials Science & Engineering

(Fe/Sr) co-doped biphasic calcium phosphate with tailored Osteoblast cell functionality Subhadip Basua, Aritri Ghoshb, Ananya Baruib,*, Bikramjit Basua,c,*

Author Address a

Laboratory for Biomaterials, Materials Research Center, Indian Institute of Science,Bangalore560012, India

b

Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur, Howrah-711103, West Bengal, India

c

Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore-560012, India

*Joint corresponding authors E-mail addresses: [email protected] (B. Basu), [email protected](A. Barui)

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Abstract Although doped bioceramics have been widely investigated for biomedical applications, the co-doped bioceramics remain mostly unexplored for bone regeneration applications. For example, the impact of co-doping of Sr2+ and Fe3+ ions on the phase stability and cytocompatibility is not explored so far. In this perspective, the objective of the present study is to quantitatively understand this aspect in case of Fe/Sr co-doped biphasic calcium phosphate (BCP).Following sol-gel synthesis, co-doped BCP samples with Sr/Fe dopant concentrations of 2, 10, 20, 30 and 40 mol%as well as doped BCP with single dopant (Sr or Fe) with similar composition were calcined at 800°C in air. Using extensive Rietveld analysis, the dopant content dependent crystallographic properties (e.g lattice parameters) and phasestability of HA/TCP are quantitatively assessed. In vitro cytocompatibility of co-doped samples has been assessed using mouse osteoblast cells. An important observation is that, while singular dopant of Sr/Fe at 20 mol% or higher amount reduces cell viability significantly, osteoblast viability is not compromised to any significant extent on Sr/Fe codoped BCP, compared to undoped BCP. Our results indicate that one can tailor osteoblast functionality by controlling the co-dopant content. More importantly, all the co-doped BCPs support cell proliferation, when single doped BCP exhibits significant reductionin cell viability, at dopant content of 10 mol% or higher. Cell morphological analysis supports extensive cell spreading on co-doped BCPs. An attempt has been made to correlate the variation in cellular response with HA/TCP ratio and ion dissolution behavior. Taken together, the present work establishes unique advantage of Sr/Fe co-doping approach towards realizing their bone replacement application. KEYWORDS:Biphasic calcium phosphate, Strontium and iron doping, Cytocompatibility, osteoblastcells.


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Introduction In the present world, musculoskeletal diseases have become a serious global issue. According to statistics, about 1.2 million people have sacrificed their life due to lack of proper bone replacement modality in clinics.1Bone loss or damage may happen due to accidents, osteoporosis or due to surgical removal of bone tissue in the course of osteosarcoma treatment.2In view of excellent osteoconductivity and osteoinductivity property, extensive research is currently underway on calcium phosphate based materials such as hydroxyapatite, tricalcium phosphate to realize better bone mimicking properties for bone replacement applications.

3, 4, 5,6

However, the major drawback of HA is its poor mechanical and functional

properties.1, 2, 3, 4Various methods have been attempted by the researchers in order to enhance the mechanical properties of HA. One such method is the isomorphic substitution. Cations (Fe2+/Fe3+, Mg2+, Zn2+, Ag2+ etc.) and anions (Cl-, F-, CO32- etc.) can substitute for Ca2+, PO43-, OH- in the crystal structure. HA has the ability to accept such substitution while retaining its crystal structure.7 These kinds of substitutions modify structure/phase assemblage and properties of Calcium Phosphate. Among various substitution, strontium and iron substitution in the HA crystal structure is important from the clinical viewpoint. Strontium plays a vital role in bone metabolism. Strontium ranelate is used as a drug for osteoporosis treatment. In case of Sr-doped HA, mixed substitution is found in both calcium sites, [Ca(1) and Ca(2)]. It is reported that, Ca(1) site is favorable for low (100 nm can be found. No definite morphology of particles could be found in any sample. The agglomerate formation has also been observed. No definite morphological change has been seen in the highly doped sample. FTIR analysis FTIR spectrum of undoped BCP has the characteristic features of that of HA (Fig. 5a). The peak at 3572 cm-1 appears due to stretching of OH- group inside the crystal. The broad peak at 3424 cm-1indicates the presence of absorbed water in the samples. An intense peak around 1041 cm-1 corresponds to v3vibrational mode of PO43- group. Also, the peaks around 567 cm-1 and 963 cm-1 correspond to v1 and v4vibrational modes of the same group, respectively. The small peak around 1465 cm-1 indicates the presence of CO32- group (impurity) inside the samples. For Sr-doped samples (Fig. 5(b)), the peaks correspond to CO32- group are present in all samples. The peak broadening of v3 vibrational mode of PO43-group is visible with an increment of dopant concentration. This may happen due to the presence of HPO42-group in the highly doped samples. Incorporation of HPO42- group in HA crystal structure with higher Sr doping is evident in the literature.50 A peak around 725 cm-1is prominent in 40 mol% Sr-doped sample (40SrBCP) and this can be attributed to the presence of P2O74- group, which is a signature of βTCP phase. It is also prominently present in 20 mol% doped sample (20SrBCP), whereas its presence is negligible in other samples. This result agrees with XRD results, where it has been found that, weight percent of β-TCP is high in two samples. In case of iron doped samples,the broad band corresponds to OH- stretching is present in all of the samples (Fig.5c). The band of CO32-decreases with increment of dopant concentration. For co-doped samples intensity of IR peak at 961 cm-1(v1 vibrational mode of PO43-) was found to decrease with increase in dopant concentration and such characteristic peak is absent in 40 mol% doped sample (20Sr20FeBCP) (Fig. 5d),. The intensity of CO32- around 1465 cm-1 also 14 ACS Paragon Plus Environment

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decreased with an increment of dopant concentration. The splitting of phosphate vibrational mode is reduced to two from three in the highly co-doped samples, showing lack of symmetry around phosphate group.

Ion dissolution, in vitro The dissolution of different ions in high glucose DMEM media with 10% HI-FBS at different time points was determined by ICPMS. Particularly, the release of calcium, strontium, phosphorus and iron ions was estimated as these ions influence cell growth. Figure 9 shows the release profile of these ions from different co-doped samples. Calcium release profile (Fig. 6(a)) shows that, for undoped and 2 mol%-doped sample (1Sr1FeBCP), concentration of Ca2+decreases with time. In contrast, for higher dopant concentration (10, 20, 30, 40 mol%), the concentration increases after the initial decrease. For phosphorus ion release profile, a similar trend is observed (Fig. 6b). For highly doped samples, the concentration of phosphorus ion increases after 48 hours. As shown in Fig. 6(c), it is found that ion concentration release becomes almost constant for 1Sr1FeBCP, after 24 hours. For highly Fe-doped BCP samples, ion concentration shows initial decreasing trend up to 48 hours and again increases. A similar behavior has been recorded in case of strontium ion release (Fig. 6(d)). In this case, ion concentration becomes almost constant for 1Sr1FeBCP and 5Sr5FeBCP doped samples and shows fluctuating behavior for rest of the samples. The above-described behavior can be explained in terms of two competing processes, dissolution and precipitation.51 Initially, dissolution rate remains higher than precipitation rate. With time, dissolution rate decreases and precipitation rate increases, until equilibrium reaches. At equilibrium, ion concentration in the solution remains unchanged with time. The decrease in ion concentration can be attributed to precipitation process. Calcium and phosphorus concurrently precipitate to form apatite layer, while the other ions may act as nucleation sites for apatite layer formation. It is well reported that the doping of strontium 15 ACS Paragon Plus Environment


increases the solubility of HA.52 Higher strontium ion release in highly doped samples corroborates well with the above phenomenon. Also, the ratio of HA to β-TCP plays an important role in ion release as β-TCP is more soluble than HA.53 Hence, ion release increases with the increment of the amount of β-TCP. In the present study, amount of β-TCP increases with an increment of dopant concentration. This is one of the probable cause behind the increase of ion release in highly-doped sample. As low amount of iron and strontium are present in low-doped samples, the equilibration takes place much rapidly. Hence, strontium and iron ion concentration become constant in 2 mol% doped sample (1Sr1FeBCP) after 24 h. During our observation period, equilibration for strontium and iron ions did not take place in highly doped samples and the ion concentration kept changing. As amount calcium and phosphorus ions are high in all of the samples, equilibrium state has not been attained for those two ions also and thus the concentration of these two ions in the solution never reached steady state. Cytocompatibility assessment Cell viability Fig. 7(a) depicts the variation of metabolically active MC3T3 cells attached on different Sr/Fe co-doped samples for a period of upto 120h in culture. One way ANOVA analysis revealed that, cell viability significantly increased only in 1Sr1FeBCP and 5Sr5FeBCP samples compared to undoped BCP after 120h of culture. Although cell viability sharply decreases in 10Sr10FeBCP and 20Sr20FeBCP, when compared to that on 5Sr5FeBCP, but not in any significant manner w.r.t undoped BCP. Therefore, Sr/Fe co-dopant upto 40 mol% does not cause any cytotoxicity to osteoblast cells.For the single doped samples,there isa significant change in cell viability in all the samples, except 2FeBCP w.r.t undoped BCP. The scenario is quite different in case of singular doped BCP. A close observation of the data in Fig. 7 reveals that with increasing the concentration of Sr and Fe in co-doped samples, the viability of osteoblast cells are significantly increased. Higher viability has been observed in 16 ACS Paragon Plus Environment

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5Sr5FeBCP (more than 200%). However, with further increase in doping concentration, a decrease in cell viability has been observed. But if we compared the cellular viability of codoped and the single-doped substrate with higher doping concentration, co-doped samples are preferred by the cell population in comparison to the single doped samples. MTT data shows that, the viability of osteoblast cells in 20Sr20FeBCP (~60%) is remarkably higher in comparison to 20SrBCP (~35%) and 20FeBCP (~50%) after 5 days in culture. The above observations confirm the fact that, although the cell viability is observed to be decreased in BCPs with higher co-dopant concentrations (20Sr20FeBCP), however co-doping at such concentration promotes significantly improved cytocompatibility in comparison to the single doped samples of similar dopant content. Live-dead assay results Fig. 8(a) -8(l) shows the representative fluorescence microscopy images after live-dead assay of the MC3T3 cells on the Sr/Fe co-doped samples and single doped samples.The quantitative analysis using imageJ software was carried out to determine the number of live and dead cells on each sample and the results are plotted in fig. 8(m) and 8(n).

In

corroboration with the MTT outcome, the live-dead data shows a significant number of live cells on undoped BCP, 1Sr1FeBCP, and 5Sr5FeBCP after 120h of culture with insignificant number of dead cells on 20Sr20FeBCP. Also, the number of live cells increase by ~1.7 fold in 5Sr5FeBCP w.r.t to undoped BCP. In case of the single doped samples,(2FeBCP, 2SrBCP & 40SrBCP)a significant number of live cells are recorded. Among single doped samples, 2SrBCPand 2FeBCPexhibithigher numbers of live cells in comparison to other groups. The number of live cells increases by ~1.5 fold and ~1.3 fold in 2FeBCP and 2SrBCP, respectively. However, in the single doped group, the number of dead cells are higher in 40SrBCP and 40FeBCP. Interestingly, similar to MTT data, a higher number of live cells on 20Sr20FeBCP are seen in comparison to the single doped samples



(40SrBCP and 40FeBCP). The above observations also indicate that the co-doped samples promote better cellular viability than single doped samples. Cell morphological analysis Fig. 9 shows the Phalloidin-DAPI staining of MC3T3 cells after 5 days of culture foreach study group. The distribution of F-actin filaments of the cellular cytoskeleton is markedly higher in 1Sr1FeBCP,5Sr5FeBCP, and 20Sr20FeBCP. In accordance with the MTT and livedead based observations, the dense osteoblast population on 2FeBCP, 2SrBCP and 40SrBCP are observed than the single doped samples. Also, Phalloidin intensity, the signature of actin filament spreading, does not show any statistically significant differences among the codoped and single doped BCPs. This observation corroborates extremely well with significant cell flattening and extensive actin reorganization on most of the investigated BCPs (Fig.10). SEM images showing better revelation of osteoblast expansion or flattening on co-doped BCPs are provided in Fig. 10. As a signatory marker of cell proliferation, the cells are seen to have well- established substrate adherence, spreading and cell-cell communication through filopodial extensions (Fig 10). Although cell viability is low on 40FeBCP/40SrBCP, we do notice cell-cell interaction and filopodial extension on these samples(Fig. 10(a), 10(i), 10(l)).

Discussion It is well known that cell viability on a material surface strongly depends on physical and chemical properties. In this section, plausible reasons behind the difference in cell viability on different co-doped BCPs have been analyzed in terms of the release profile of different ions and phase composition. In conventional cell biology experiments, the various growth factors are added to culture medium and their influence, together with or without a drug, is assessed on osteoblast cells. In case of bioresorbable ceramics, released ions in the cell culture medium play a vital role in influencing cell proliferation and differentiation. In particular, the role of Ca, P Sr and Fe


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ions, in the present case, are noteworthy in osteoblastic growth regulation and osteogenic differentiation. Being the main component of bioapatite, Ca and P both have an important role in bone formation and resorption. Ca is known to affect osteoblast cells, in vitro. Low (2-4 mmol) and medium (6-8 mmol) Ca2+ concentration is suitable for osteoblastic proliferation, differentiation and extracellular matrix remineralisation, respectively.54Also, Ca2+ ions take part in bone remineralisation by directly activating intercellular mechanisms via Ca-ion channels (L and non L type) and Ca-sensing cellular receptors.55It is also reported that, Ca2+ regulates osteoblast proliferation by regulating the expression of growth factors like IGF-I and IGF-II.56 Extracellular Ca increases glutamate release of osteoblast cells and thus regulating bone mechanosensitivity.57In a different study, it is reported that, extracellular Ca may be internalized via ion channels and thereafter activate CAMK2α/CAM pathway,58which can potentially modulate osteoblastic differentiation via CREB/ERK1/2 dependent pathways.59The increased Ca2+ levels in cell culture medium enhanced the expression of BMP-2 mRNA in hBMSCs via activating PKC and ERK1/2dependent pathways.60 In spite of the above beneficiary roles, excess Ca2+ concentrations (>10mmol) are cytotoxic and may induce apoptosis.55In the present case, Ca2+ ion dissolution is not significant among all the co-doped samples and decreases with time upto 72 hours at lowly-doped samples (undoped and 1Sr1FeBCP). Therefore, the difference in cellular response can be explained on the basis of Ca2+dissolution. In contrast, P appears to play secondary role in osteoblastic proliferation and differentiation.61For example, P ion stimulates expression of matrix Gla protein, a regulator of bone formation in osteoblast cells.62 Specific induction of OPN in MC3T3-E1 cells due to the increased level of P has been reported in the literature.63The elevation of OPN takes place via activation of PKC and ERK/2 dependent pathways.64 However, the presence of Ca2+ is required for P to activate ERK1/2 dependent pathways via the formation of calcium 19 ACS Paragon Plus Environment


phosphate precipitate outside the cell.65 Hence, P only plays a minor role in osteoblastic differentiation. It has been reported that, while 0.09mM of P supports osteoblast proliferation, concentration higher than that leads to significant cell apoptosis.66 On the other hand, because of chemical similarity, Sr2+ can accumulate in bone by exchanging with Ca2+.67Also, Sr2+ plays beneficial roles on bone by enhancing osteoblastic activity and suppressing osteoclastic activity.68, 69Sr2+ ion concentration around 10-3 M has been reported to favor bone formation.70Sr2+ can enhance osteogenic differentiation by promoting transcriptional activity of RUNX2 via activation of ERK1/2 and P38 dependent pathways.71Apart from that, Sr2+concentration around 0.2107 mg/l is known topromote osteoblastic differentiation of hMSC via inducing early expression of Cbf1 gene.72 However, high concentration of Sr in cell culture media can cause adverse effect on cells.73 Reduction in matrix mineralization is found at high (20mg/l) concentration of Sr.74Likewise, the therapeutic benefits of Fe3+ are implicated in the increased risk of osteoporosis due to iron deficiency as well as in calcium homeostasis in healthy bone and teeth.75 Fe-doped HA is known to promote osteoblastic proliferation.25An excess amount of Fe (0.1 to 1.0 mg/l) is reported to inhibit osteoblast proliferation and differentiation. 76Besides that, high amount of Fe3+can also lead to cell apoptosis via suppression of ALP activity and increment of ROS formation.77,78Consistent with the above, we now analyze how ion release profoundly influences the osteoblastic behavior in the present case. From fig. 6(a), it is clearly seen that, the concentration of Ca2+varies between 3.3 mg/l and 4.0 mg/l (at 72h), which is favorable for osteoblastic proliferation and differentiation. Also, the amount of P is also recorded to be within the tolerable limit (0.4 mg/l to 0.9 mg/l at 72h) for osteoblast cell growth (see Fig. 6(b)).On the other hand, concentration of Sr2+is found to vary between 0.009 mg/l & 0.196 mg/l whereas that of Fe3+varies between 0.023 mg/l & 0.074 mg/l (Fig. 6(c) and 6(d)). The significant increase in cell viability in 1Sr1FeBCP and 5Sr5FeBCP compared to undoped BCP can be attributed to the beneficiary roles of Sr2+ and Fe3+ions in the culture media, in 20 ACS Paragon Plus Environment

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parallel with Ca2+. In Fig. 6, a clear increasing trend of Sr2+release with an increase of dopant content is visible.

Hence, Sr2+ concentration is high in culture media in case of

20Sr20FeBCP. Although high concentration of Sr2+has some adverse effect on osteoblast cells, insignificant change in cell viability in 20Sr20FeBCP w.r.t to undoped sample rules out such effect in case of co-doped samples. From the live dead and DAPI-Phalloidin images, significant number of live, healthy and well-spread osteoblast cells are visible on 20Sr20FeBCP. Hence, it can be concluded that, in spite of insignificant changes in cell viability, 20Sr20FeBCP certainly does not have any toxic effect on osteoblast cells. The same is true for 40SrBCP. Decrease in cell viability observed for10Se10FeBCP may be attributed to high Fe3+release in culture media. It can be clearly seen in Fig. 6(d) that, the amount of Fe3+released from this sample (0.074 mg/l at 72h) is quite close to the toxic limit (0.1mg/l). Probably due to the same reason, cell viability also decreases in case of 40FeBCP. However, these samples also cannot be distinguished as cytotoxic as significant numbers of live cells can be found on these samples(Fig. 8d and 8l). Apart from ion release, phase abundance of β-TCP and HA plays an important role in regulating cell viability and functionality. As mentioned before, β-TCP is more soluble than stable HA, in vitro.53With the increment ofβ-TCP phase, the solubility and thus calcium ion release increases and affects cell viability. A correlation between Fig.3 and Fig. 7(a) can be used to substantiate this point further. Fig. 3 reveals a moderate increase in β-TCP with Sr/Fe dopant upto 10 mol%. While β-TCP wt% remains similar for the increase in (Sr/Fe) codopant in the range of 10-30 mol%, a sharp increase is recorded for an increase from 30 to 40 mol%, leading to more solubility. For Sr and Fe-doped samples, HA and β-TCP remain the major phase, respectively. In 40FeBCP, β-TCP content quite high leading to a release of a high amount of Fe3+ in culture media. At the closure, the contrasting influence of single doped vs. co-doped BCPs on cell viability is depicted in Fig. 11. While ion release has been shown to regulate cell proliferation, one has to 21 ACS Paragon Plus Environment


also remember the difference in phase abundance and its potential effect on osteoblast viability. Also, the collective effect of dissolution of different ions in co-doped samples appears to favor osteoblast growth on BCPs doped with Sr/Fe upto 40 mol%, while singular dopant of similar content causes significant reduction in cell viability.

Conclusions Sol-gel method has been successfully employed in the present study to synthesize ultrafine (submicron) Sr/Fe co-doped BCP. The structural changes in terms of crystallographic lattice parameter variant and phase stability are quantified using XRD based Rietveld refinement in a reliable manner. The following conclusions can be drawn from different aspects of the present study. a)

In comparison to Sr-doped BCP, Fe-doped BCPs exhibit a systematic increase in β-

TCP content with dopant amount. In contrast, HA remains the major phase in case of Srdoped BCPs. In case of co-doped BCPs, the competing effect of Fe3+ and Sr2+ dopant is reflected in gradual increase of β-TCP phase content with destabilizing effect at higher codopant content. b)

The structural changes in terms of lattice expansion are significant in (Sr/Fe) co-

doped BCPs. While lattice parameter changes go through systematic variation in Sr-doped BCP, such variation is rather abrupt in case of Fe-doped BCP. c)

In case of singular doped BCPs, higher dopant content appears to reduce osteoblast

cell viability. However, immunofluorescence staining reveals healthy cell morphology on all the investigated BCPs. d)

The competition among HA/TCP ratio and Fe3+/Sr2+/Ca2+ ion dissolution has

significant influence on osteoblast cell functionality on co-doped BCPs. While co-doped


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BCP with 5 mol% Sr and 5 mol% Fe-dopant exhibits highest cell viability, a decrease is recorded at higher co-dopant content, but not to any significant extent w.r.t. undoped BCP. In summary, our study has established Sr/Fe co-doped samples as a suitable candidate for bone replacement applications. Overall, the present study clearly establishes a rational approach for the design of next generation of doped bioactive ceramics.

Supporting Information available The following files are available free of charge. Supplementary document.docx: Contains XRD images of undoped and different kind of doped samples.

Acknowledgements The authors want to acknowledge the Department of Science and technology (DST) for their financial support. We thank Centre for Nano Science and Engineering (CenSE), IISc for FTIR data recording. The authors (AG and AB)also thank TEQIP II, IIEST Shibpur for the Nikon Microscopy facility.



References: 1. Basu, B., BIomaterials Science and Tissue Engineering: Principles and Methods; . Cambridge University Press: 2017. 2. Basu, B., Biomaterials for Musculoskeletal Regeneration: Concepts. Springer: 2016. DOI: 10.1007/978-981-10-3059-8. 3. Basu, B.; Katti, D. S.; Kumar, A., Advanced biomaterials: fundamentals, processing, and applications. John Wiley & Sons: 2010. DOI: 10.1002/9780470891315. 4. Basu, B.; Ghosh, S., Biomaterials for Musculoskeletal Regeneration: Applications. Springer Singapore: 2016. DOI: 10.1007/978-981-10-3017-8. 5. Kazemzadeh Narbat, M.; Orang, F.; Solati Hashtjin, M.; Goudarzi, A., Fabrication of porous hydroxyapatite-gelatin composite scaffolds for bone tissue engineering. Iranian Biomedical Journal 2006, 10 (4), 215-223. 6. Komlev, V. S.; Barinov, S. M.; Koplik, E. V., A method to fabricate porous spherical hydroxyapatite granules intended for time-controlled drug release. Biomaterials 2002, 23 (16), 34493454. DOI: https://doi.org/10.1016/S0142-9612(02)00049-2. 7. Matsumura, Y.; Moffat, J. B., Methanol adsorption and dehydrogenation over stoichiometric and non-stoichiometric hydroxyapatite catalysts. Journal of the Chemical Society, Faraday Transactions 1996, 92 (11), 1981-1984. DOI: https://doi.org/10.1039/ft9969201981. 8. Terra, J.; Dourado, E. R.; Eon, J.-G.; Ellis, D. E.; Gonzalez, G.; Rossi, A. M., The structure of strontium-doped hydroxyapatite: an experimental and theoretical study. Physical Chemistry Chemical Physics 2009, 11 (3), 568-577. DOI: https://doi.org/10.1039/B802841A. 9. Marie, P., Strontium ranelate: a novel mode of action optimizing bone formation and resorption. Osteoporosis International 2005, 16 (1), S7-S10. DOI: https://doi.org/10.1007/s00198004-1753-8. 10. Bonnelye, E.; Chabadel, A.; Saltel, F.; Jurdic, P., Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro. Bone 2008, 42 (1), 129-138. DOI: https://doi.org/10.1016/j.bone.2007.08.043. 11. Marie, P. J.; Hott, M.; Modrowski, D.; De Pollak, C.; Guillemain, J.; Deloffre, P.; Tsouderos, Y., An uncoupling agent containing strontium prevents bone loss by depressing bone resorption and maintaining bone formation in estrogen-deficient rats. Journal of Bone and Mineral Research 2005, 20 (6), 1065-1074. DOI: https://doi.org/10.1359/jbmr.2005.20.6.1065. 12. Dahl, S.; Allain, P.; Marie, P.; Mauras, Y.; Boivin, G.; Ammann, P.; Tsouderos, Y.; Delmas, P.; Christiansen, C., Incorporation and distribution of strontium in bone. Bone 2001, 28 (4), 446-453. DOI: https://doi.org/10.1016/S8756-3282(01)00419-7. 13. Sabareeswaran, A.; Basu, B.; Shenoy, S. J.; Jaffer, Z.; Saha, N.; Stamboulis, A., Early osseointegration of a strontium containing glass ceramic in a rabbit model. Biomaterials 2013, 34 (37), 9278-9286. DOI: https://doi.org/10.1016/j.biomaterials.2013.08.070. 14. Basu, B.; Sabareeswaran, A.; Shenoy, S. J., Biocompatibility property of 100% strontiumsubstituted SiO2–Al2O3–P2O5–CaO–CaF2 glass ceramics over 26 weeks implantation in rabbit model: Histology and micro-Computed Tomography analysis. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2015, 103 (6), 1168-1179. DOI: 10.1002/jbm.b.33270. 15. Capuccini, C.; Torricelli, P.; Sima, F.; Boanini, E.; Ristoscu, C.; Bracci, B.; Socol, G.; Fini, M.; Mihailescu, I.; Bigi, A., Strontium-substituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response. Acta Biomaterialia 2008, 4 (6), 1885-1893. DOI: https://doi.org/10.1016/j.actbio.2008.05.005. 16. Gentleman, E.; Fredholm, Y. C.; Jell, G.; Lotfibakhshaiesh, N.; O'Donnell, M. D.; Hill, R. G.; Stevens, M. M., The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro. Biomaterials 2010, 31 (14), 3949-3956. DOI: https://doi.org/10.1016/j.biomaterials.2010.01.121. 17. Isaac, J.; Nohra, J.; Lao, J.; Jallot, E.; Nedelec, J.-M.; Berdal, A.; Sautier, J.-M., Effects of strontium-doped bioactive glass on the differentiation of cultured osteogenic cells. Eur Cell Mater 2011, 21, 130-43. DOI: https://doi.org/10.22203/eCM.v021a11. 24 ACS Paragon Plus Environment

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18. Capuccini, C.; Torricelli, P.; Boanini, E.; Gazzano, M.; Giardino, R.; Bigi, A., Interaction of Sr-doped hydroxyapatite nanocrystals with osteoclast and osteoblast-like cells. Journal of Biomedical Materials Research Part A 2009, 89 (3), 594-600. 19. Sabareeswaran, A.; Basu, B.; Shenoy, S. J.; Jaffer, Z.; Saha, N.; Stamboulis, A., Early osseointegration of a strontium containing glass ceramic in a rabbit model. Biomaterials 2013, 34 (37), 9278-9286. DOI: https://doi.org/10.1016/j.biomaterials.2013.08.070. 20. Krishnan, V.; Bhatia, A.; Varma, H., Development, characterization and comparison of two strontium doped nano hydroxyapatite molecules for enamel repair/regeneration. Dental Materials 2016, 32 (5), 646-659. DOI: https://doi.org/10.1016/j.dental.2016.02.002. 21. Stojanović, Z.; Veselinović, L.; Marković, S.; Ignjatović, N.; Uskoković, D., Hydrothermal synthesis of nanosized pure and cobalt-exchanged hydroxyapatite. Materials and Manufacturing Processes 2009, 24 (10-11), 1096-1103. DOI: https://doi.org/10.1080/10426910903032113. 22. Tampieri, A.; D’Alessandro, T.; Sandri, M.; Sprio, S.; Landi, E.; Bertinetti, L.; Panseri, S.; Pepponi, G.; Goettlicher, J.; Bañobre-López, M., Intrinsic magnetism and hyperthermia in bioactive Fe-doped hydroxyapatite. Acta biomaterialia 2012, 8 (2), 843-851. DOI: https://doi.org/10.1016/j.actbio.2011.09.032. 23. Ajeesh, M.; Francis, B. F.; Annie, J.; Harikrishna Varma, P. R., Nano iron oxide–hydroxyapatite composite ceramics with enhanced radiopacity. Journal of Materials Science: Materials in Medicine 2010, 21 (5), 1427-1434. DOI: 10.1007/s10856-010-4005-9. 24. Wu, H.-C.; Wang, T.-W.; Sun, J.-S.; Wang, W.-H.; Lin, F.-H., A novel biomagnetic nanoparticle based on hydroxyapatite. Nanotechnology 2007, 18 (16), 165601. DOI: https://doi.org/10.1088/0957-4484/18/16/165601. 25. Sarath Chandra, V.; Baskar, G.; Suganthi, R.; Elayaraja, K.; Ahymah Joshy, M.; Sofi Beaula, W.; Mythili, R.; Venkatraman, G.; Narayana Kalkura, S., Blood compatibility of iron-doped nanosize hydroxyapatite and its drug release. ACS applied materials & interfaces 2012, 4 (3), 1200-1210. DOI: https://doi.org/10.1021/am300140q. 26. Jiang, M.; Terra, J.; Rossi, A.; Morales, M.; Saitovitch, E. B.; Ellis, D., Fe 2+/F e 3+ substitution in hydroxyapatite: Theory and experiment. Physical Review B 2002, 66 (22), 224107. DOI: https://doi.org/10.1103/PhysRevB.66.224107. 27. Boda, S. K.; Basu, B.; Sahoo, B., Structural and magnetic phase transformations of hydroxyapatite-magnetite composites under inert and ambient sintering atmospheres. The Journal of Physical Chemistry C 2015, 119 (12), 6539-6555. DOI: https://doi.org/10.1021/jp5114027. 28. Matsumoto, N.; Sato, K.; Yoshida, K.; Hashimoto, K.; Toda, Y., Preparation and characterization of β-tricalcium phosphate co-doped with monovalent and divalent antibacterial metal ions. Acta Biomaterialia 2009, 5 (8), 3157-3164. DOI: https://doi.org/10.1016/j.actbio.2009.04.010. 29. Quillard, S.; Paris, M.; Deniard, P.; Gildenhaar, R.; Berger, G.; Obadia, L.; Bouler, J.-M., Structural and spectroscopic characterization of a series of potassium-and/or sodium-substituted βtricalcium phosphate. Acta biomaterialia 2011, 7 (4), 1844-1852. DOI: https://doi.org/10.1016/j.actbio.2010.12.016. 30. Vahabzadeh, S.; Hack, V. K.; Bose, S., Lithium-doped β-tricalcium phosphate: Effects on physical, mechanical and in vitro osteoblast cell–material interactions. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2017, 105 (2), 391-399. DOI: https://doi.org/10.1002/jbm.b.33485. 31. Kim, T.-W.; Lee, H.-S.; Kim, D.-H.; Jin, H.-H.; Hwang, K.-H.; Lee, J. K.; Park, H.-C.; Yoon, S.-Y., In situ synthesis of magnesium-substituted biphasic calcium phosphate and in vitro biodegradation. Materials Research Bulletin 2012, 47 (9), 2506-2512. DOI: https://doi.org/10.1016/j.materresbull.2012.05.011. 32. Kaur, G.; Pandey, O. P.; Singh, K.; Homa, D.; Scott, B.; Pickrell, G., A review of bioactive glasses: their structure, properties, fabrication and apatite formation. Journal of Biomedical Materials Research Part A 2014, 102 (1), 254-274. DOI: https://doi.org/10.1002/jbm.a.34690.



33. Dorozhkin, S. V.; Epple, M., Biological and medical significance of calcium phosphates. Angewandte Chemie International Edition 2002, 41 (17), 3130-3146. DOI: https://doi.org/10.1002/1521-3773(20020902)41:173.0.CO;2-1. 34. Kaygili, O.; Dorozhkin, S. V.; Ates, T.; Al-Ghamdi, A. A.; Yakuphanoglu, F., Dielectric properties of Fe doped hydroxyapatite prepared by sol–gel method. Ceramics International 2014, 40 (7), 93959402. DOI: https://doi.org/10.1016/j.ceramint.2014.02.009. 35. Larson, A. C.; Von Dreele, R. B., Gsas. General Structure Analysis System. LANSCE, MS-H805, Los Alamos, New Mexico 1994. 36. Boda, S. K.; Thrivikraman, G.; Panigrahy, B.; Sarma, D. D.; Basu, B., Competing Roles of Substrate Composition, Microstructure, and Sustained Strontium Release in Directing Osteogenic Differentiation of hMSCs. ACS Applied Materials & Interfaces 2017, 9 (23), 19389-19408. DOI: 10.1021/acsami.6b08694. 37. Chartier, A.; Meis, C.; Gale, J. D., Computational study of Cs immobilization in the apatites Ca 10 (PO 4) 6 F 2, Ca 4 La 6 (SiO 4) 6 F 2 and Ca 2 La 8 (SiO 4) 6 O 2. Physical Review B 2001, 64 (8), 085110. DOI: https://doi.org/10.1103/PhysRevB.64.085110. 38. Meis, C.; Gale, J.; Boyer, L.; Carpena, J.; Gosset, D., Theoretical study of Pu and Cs incorporation in a mono-silicate neodymium fluoroapatite Ca9Nd (SiO4)(PO4) 5F2. The Journal of Physical Chemistry A 2000, 104 (22), 5380-5387. DOI: https://doi.org/10.1021/jp000096j. 39. Verberckmoes, S.; Behets, G.; Oste, L.; Bervoets, A.; Lamberts, L.; Drakopoulos, M.; Somogyi, A.; Cool, P.; Dorrine, W.; De Broe, M., Effects of strontium on the physicochemical characteristics of hydroxyapatite. Calcified tissue international 2004, 75 (5), 405-415. DOI: https://doi.org/10.1007/s00223-004-0260-4. 40. Kim, H.-W.; Koh, Y.-H.; Kong, Y.-M.; Kang, J.-G.; Kim, H.-E., Strontium substituted calcium phosphate biphasic ceramics obtained by a powder precipitation method. Journal of Materials Science: Materials in Medicine 2004, 15 (10), 1129-1134. DOI: 10.1023/b:jmsm.0000046395.76435.60. 41. Renaudin, G.; Jallot, E.; Nedelec, J.-M., Effect of strontium substitution on the composition and microstructure of sol–gel derived calcium phosphates. Journal of sol-gel science and technology 2009, 51 (3), 287-294. DOI: https://doi.org/10.1007/s10971-008-1854-5. 42. Bigi, A.; Boanini, E.; Capuccini, C.; Gazzano, M., Strontium-substituted hydroxyapatite nanocrystals. Inorganica Chimica Acta 2007, 360 (3), 1009-1016. DOI: http://dx.doi.org/10.1016/j.ica.2006.07.074. 43. Kavitha, M.; Subramanian, R.; Narayanan, R.; Udhayabanu, V., Solution combustion synthesis and characterization of strontium substituted hydroxyapatite nanocrystals. Powder technology 2014, 253, 129-137. DOI: https://doi.org/10.1016/j.powtec.2013.10.045. 44. Mardziah, C.; Sopyan, I.; Ramesh, S., Strontium-doped hydroxyapatite nanopowder via solgel method: effect of strontium concentration and calcination temperature on phase behavior. Artif. Organs 2009, 23 (2), 105-113. 45. Ryu, H.-S.; Hong, K. S.; Lee, J.-K.; Kim, D. J., Variations of structure and composition in magnesium incorporated hydroxyapatite/β-tricalcium phosphate. Journal of materials research 2006, 21 (2), 428-436. DOI: https://doi.org/10.1557/jmr.2006.0049. 46. Wang, J.; Nonami, T.; Yubata, K., Syntheses, structures and photophysical properties of iron containing hydroxyapatite prepared by a modified pseudo-body solution. Journal of Materials Science: Materials in Medicine 2008, 19 (7), 2663-2667. DOI: https://doi.org/10.1007/s10856-0073365-2. 47. Anee, T.; Palanichamy, M.; Ashok, M.; Sundaram, N. M.; Kalkura, S. N., Influence of iron and temperature on the crystallization of calcium phosphates at the physiological pH. Materials Letters 2004, 58 (3), 478-482. DOI: https://doi.org/10.1016/S0167-577X(03)00529-9. 48. Morrissey, R.; Rodriguez-Lorenzo, L.; Gross, K., Influence of ferrous iron incorporation on the structure of hydroxyapatite. Journal of Materials Science: Materials in Medicine 2005, 16 (5), 387392. DOI: https://doi.org/10.1007/s10856-005-6976-5.


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49. Low, H.; Phonthammachai, N.; Maignan, A.; Stewart, G.; Bastow, T.; Ma, L.; White, T., The crystal chemistry of ferric oxyhydroxyapatite. Inorganic chemistry 2008, 47 (24), 11774-11782. DOI: https://doi.org/10.1021/ic801491t. 50. Landi, E.; Sprio, S.; Sandri, M.; Celotti, G.; Tampieri, A., Development of Sr and CO 3 cosubstituted hydroxyapatites for biomedical applications. Acta Biomaterialia 2008, 4 (3), 656-663. DOI: https://doi.org/10.1016/j.actbio.2007.10.010. 51. Ereiba, K. M. T.; Mostafa, A.; Gamal, G.; Said, A., In vitro study of iron doped hydroxyapatite. Journal of Biophysical Chemistry 2013, 4 (4), 122. DOI: https://doi.org/10.4236/jbpc.2013.44017. 52. Pan, H.; Li, Z.; Lam, W.; Wong, J.; Darvell, B.; Luk, K.; Lu, W., Solubility of strontiumsubstituted apatite by solid titration. Acta Biomaterialia 2009, 5 (5), 1678-1685. DOI: https://doi.org/10.1016/j.actbio.2008.11.032. 53. Lobo, S. E.; Livingston Arinzeh, T., Biphasic calcium phosphate ceramics for bone regeneration and tissue engineering applications. Materials 2010, 3 (2), 815-826. DOI: https://doi.org/10.3390/ma3020815. 54. Maeno, S.; Niki, Y.; Matsumoto, H.; Morioka, H.; Yatabe, T.; Funayama, A.; Toyama, Y.; Taguchi, T.; Tanaka, J., The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture. Biomaterials 2005, 26 (23), 4847-4855. DOI: https://doi.org/10.1016/j.biomaterials.2005.01.006. 55. Hoppe, A.; Güldal, N. S.; Boccaccini, A. R., A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011, 32 (11), 27572774. DOI: https://doi.org/10.1016/j.biomaterials.2011.01.004. 56. Valerio, P.; Pereira, M.; Goes, A.; Leite, M. F., Effects of extracellular calcium concentration on the glutamate release by bioactive glass (BG60S) preincubated osteoblasts. Biomedical Materials 2009, 4 (4), 045011. DOI: https://doi.org/10.1088/1748-6041/4/4/045011. 57. Hinoi, E.; Takarada, T.; Yoneda, Y., Glutamate signaling system in bone. Journal of pharmacological sciences 2004, 94 (3), 215-220. DOI: https://doi.org/10.1254/jphs.94.215. 58. Jung, G.-Y.; Park, Y.-J.; Han, J.-S., Effects of HA released calcium ion on osteoblast differentiation. Journal of Materials Science: Materials in Medicine 2010, 21 (5), 1649-1654. DOI: https://doi.org/10.1007/s10856-010-4011-y. 59. Zayzafoon, M.; Fulzele, K.; McDonald, J. M., Calmodulin and calmodulin-dependent kinase IIα regulate osteoblast differentiation by controlling c-fos expression. Journal of Biological Chemistry 2005, 280 (8), 7049-7059. DOI: https://doi.org/10.1074/jbc.M412680200. 60. Barradas, A. M.; Fernandes, H. A.; Groen, N.; Chai, Y. C.; Schrooten, J.; van de Peppel, J.; van Leeuwen, J. P.; van Blitterswijk, C. A.; de Boer, J., A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells. Biomaterials 2012, 33 (11), 3205-3215. DOI: https://doi.org/10.1016/j.biomaterials.2012.01.020. 61. Samavedi, S.; Whittington, A. R.; Goldstein, A. S., Calcium phosphate ceramics in bone tissue engineering: a review of properties and their influence on cell behavior. Acta biomaterialia 2013, 9 (9), 8037-8045. DOI: https://doi.org/10.1016/j.actbio.2013.06.014. 62. Julien, M.; Khoshniat, S.; Lacreusette, A.; Gatius, M.; Bozec, A.; Wagner, E. F.; Wittrant, Y.; Masson, M.; Weiss, P.; Beck, L., Phosphate-Dependent Regulation of MGP in Osteoblasts: Role of ERK1/2 and Fra-1. Journal of Bone and Mineral Research 2009, 24 (11), 1856-1868. DOI: https://doi.org/10.1359/jbmr.090508. 63. Beck, G. R.; Zerler, B.; Moran, E., Phosphate is a specific signal for induction of osteopontin gene expression. Proceedings of the National Academy of Sciences 2000, 97 (15), 8352-8357. DOI: https://doi.org/10.1073/pnas.140021997. 64. Beck, G. R.; Knecht, N., Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasome-dependent. Journal of Biological Chemistry 2003, 278 (43), 41921-41929. DOI: https://doi.org/10.1074/jbc.M304470200. 65. Khoshniat, S.; Bourgine, A.; Julien, M.; Petit, M.; Pilet, P.; Rouillon, T.; Masson, M.; Gatius, M.; Weiss, P.; Guicheux, J., Phosphate-dependent stimulation of MGP and OPN expression in osteoblasts via the ERK1/2 pathway is modulated by calcium. Bone 2011, 48 (4), 894-902. DOI: https://doi.org/10.1016/j.bone.2010.12.002. 27 ACS Paragon Plus Environment


66. Liu, Y. K.; Lu, Q. Z.; Pei, R.; Ji, H. J.; Zhou, G. S.; Zhao, X. L.; Tang, R. K.; Zhang, M., The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: implication for bone tissue engineering. Biomedical materials 2009, 4 (2), 025004. DOI: https://doi.org/10.1088/1748-6041/4/2/025004. 67. Boivin, G.; Deloffre, P.; Perrat, B.; Panczer, G.; Boudeulle, M.; Mauras, Y.; Allain, P.; Tsouderos, Y.; Meunier, P. J., Strontium distribution and interactions with bone mineral in monkey iliac bone after strontium salt (S 12911) administration. Journal of Bone and Mineral Research 1996, 11 (9), 1302-1311. DOI: https://doi.org/10.1002/jbmr.5650110915. 68. Marie, P.; Ammann, P.; Boivin, G.; Rey, C., Mechanisms of action and therapeutic potential of strontium in bone. Calcified tissue international 2001, 69 (3), 121-129. DOI: https://doi.org/10.1007/s002230010055. 69. Marie, P., Strontium ranelate: a physiological approach for optimizing bone formation and resorption. Bone 2006, 38 (2), 10-14. DOI: https://doi.org/10.1016/j.bone.2005.07.029. 70. Canalis, E.; Hott, M.; Deloffre, P.; Tsouderos, Y.; Marie, P., The divalent strontium salt S12911 enhances bone cell replication and bone formation in vitro. Bone 1996, 18 (6), 517-523. DOI: https://doi.org/10.1016/8756-3282(96)00080-4. 71. Xiao, G.; Jiang, D.; Thomas, P.; Benson, M. D.; Guan, K.; Karsenty, G.; Franceschi, R. T., MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. Journal of Biological Chemistry 2000, 275 (6), 4453-4459. DOI: https://doi.org/10.1074/jbc.275.6.4453. 72. Sila-Asna, M.; Bunyaratvej, A.; Maeda, S.; Kitaguchi, H.; Bunyaratavej, N., Osteoblast differentiation and bone formation gene expression in strontium-inducing bone marrow mesenchymal stem cell. Kobe J Med Sci 2007, 53 (1-2), 25-35. 73. Schumacher, M.; Lode, A.; Helth, A.; Gelinsky, M., A novel strontium (II)-modified calcium phosphate bone cement stimulates human-bone-marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation in vitro. Acta biomaterialia 2013, 9 (12), 9547-9557. DOI: https://doi.org/10.1016/j.actbio.2013.07.027. 74. Verberckmoes, S. C.; De Broe, M. E.; D'Haese, P. C., Dose-dependent effects of strontium on osteoblast function and mineralization. Kidney International 2003, 64 (2), 534-543. DOI: https://doi.org/10.1046/j.1523-1755.2003.00123.x. 75. Toxqui, L.; Vaquero, M. P., Chronic iron deficiency as an emerging risk factor for osteoporosis: a hypothesis. Nutrients 2015, 7 (4), 2324-2344. DOI: https://doi.org/10.3390/nu7042324. 76. Yamasaki, K.; Hagiwara, H., Excess iron inhibits osteoblast metabolism. Toxicology Letters 2009, 191 (2), 211-215. DOI: https://doi.org/10.1016/j.toxlet.2009.08.023. 77. Crichton, R. R.; Boelaert, J. R., Inorganic biochemistry of iron metabolism: from molecular mechanisms to clinical consequences. John Wiley & Sons: 2001. DOI: https://doi.org/10.1002/0470845791. 78. He, Y.-F.; Ma, Y.; Gao, C.; Zhao, G.-y.; Zhang, L.-L.; Li, G.-F.; Pan, Y.-Z.; Li, K.; Xu, Y.-J., Iron overload inhibits osteoblast biological activity through oxidative stress. Biological trace element research 2013, 152 (2), 292-296. DOI: https://doi.org/10.1007/s12011-013-9605-z.


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List of figures

(a)

(b)

(c) Fig. 1: Type and amount of doping has significant influence on phase evolution in doped BCPs. Peak shift in XRD pattern of (a) Sr-doped samples, (b) Fe-doped samples and (c) Sr-Fe co-doped samples. HA Peak shifts towards smaller 2θ angle is a signature of doping inside HA lattice.



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(b) β-TCP (a)HA

(d) β-TCP

(c) HA

(e) HA

(f) β-TCP

Fig 2: HA/TCP lattice parameters show significant changes, depending on type and amount of dopant. Graphical representation of changes in lattice parameters of Sr-doped (a) HA and (b) β-TCP; Fe-doped (c) HA and (d) β-TCP and Sr/Fe co-doped (e) HA and (f) β-TCP. Change in lattice parameters is an evidence of successful doping inside crystal structure. 30 ACS Paragon Plus Environment

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(b)

(a)

(c)

Fig. 3: The phase abundance of HA and TCP in doped BCPs is significantly affected by Sr/Fe doping. Change in weight percentage of HA and β-TCP for (a) Sr-doped (b) Fe-doped, (c) Sr-Fe co-doped powder samples with dopant content.



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(a)

(b)

Fig 4: HA/TCP particle morphology is unaltered. Bright field TEM images of (a) 20SrBCP and (b) 40SrBCP, revealing the absence of any definite morphology. (Sample designation: xSryFeBCP means x mol% Sr y mol% Fe codoped biphasic calcium phosphate.) 32 ACS Paragon Plus Environment

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(b) (a)

(d)

(c)

FIG 5: Doping does not significantly affect vibrational activity of different bonds, FTIR spectrum of (a) undoped, (b) Sr-doped, (c) Fe-doped and (d) Sr-Fe co-doped powder samples.



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(b)

(a)


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(c)

(d)

Fig. 6: Dopant type and amount has a significant effect on ion dissolution. Presence of ions in culture media affects cell behavior. Elemental concentrations of (a) Ca, (b) P, (c) Fe and (d) Sr released into high glucose DMEM media with 10% HI-FBS from different co-doped samples was measured by ICPMS. All the experiments were carried out in triplicates and data shown corresponds to mean ± sd of three measurements from three individual experiments. (Sample designation: xSryFeBCP means x mol% Sr y mol% Fe co-doped biphasic calcium phosphate.)



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(a)

(b) Fig.7:Dopant type and amount influenceosteoblast cell viability in a significant manner and viability changes with dopant concentration. In order to perform quantitative assessment of cytocompatibility of different samples, MTT assay of osteoblast cells attached on different (a) Sr/Fe co-doped and (b) Sr/Fe single-doped samples on day 5 was performed. Along x axis, the percentage of co-doping is indicated. Statistical difference in cell viability is revealed by one-way ANOVA. *and** indicate statistically significant difference at P< 0.05and P

Sr) Codoped Biphasic Calcium Phosphate with Tailored

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