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Biocompatible β-SrHPO4 clusters with dandelion-like structure as an alternative drug carrier
Materials Science & Engineering C 81 (2017) 8–12
Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Short communication
Biocompatible β-SrHPO4 clusters with dandelion-like structure as an alternative drug carrier
MARK
Haishan Shia,b,c, Tingting Wua,b,c, Jing Zhangb,c, Xiaoling Yeb,c, Shenghui Zenga, Xu Liua, Tao Yua,⁎, Jiandong Yeb,c,⁎⁎, Changren Zhoua a b c
College of Chemistry and Materials, Jinan University, Guangzhou 510632, PR China School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, PR China National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Strontium hydrogen phosphate Calcium phosphate Bone mesenchymal stem cells
Recent researches about calcium phosphate (CaP) biomaterials used as drug delivery systems are focusing on the better understanding of the microenvironment around the implant-host tissue interface, with the aim to provide a bone response in pathological ones. Towards the improvement of the osteogenic potential of CaP drug carriers, dandelion-like β-SrHPO4 clusters (Φ10–20 μm) has been prepared by a homogeneous precipitation method under the hydrolysis of carbamide. Adhesion, spreading, proliferation, osteogenic differentiation and mRNA expression of bone mesenchymal stem cells (BMSCs) mediated by β-SrHPO4 clusters were investigated. Highly osteoconductive and biodegradable octacalcium phosphate with similar structure was employed as the control. By contrast, β-SrHPO4 clusters exhibited remarkably better affinity, enhanced proliferation and osteogenic differentiation of BMSCs, providing a promising alternative bioactive bone substitute and drug carrier for tissue repair. With the unique dandelion-like microstructure, we believe that our as-prepared material will open up new avenues for applicability of CaP drug delivery systems in the near future.
1. Introduction Recently, hybrid materials and inorganic materials are widely recognized as upcoming materials for delivery, therapy, diagnosis, imaging, sensors, etc. in optical, electrical, biomedical, medical, clinical and other applications [1–12]. With the compositional similarities to bone minerals and excellent biocompatibility, calcium phosphates (CaPs) are widely used in bone regeneration. CaPs have become the research focus in simultaneous use as bone substitute and drug delivery vehicle [13]. As drug carriers, the biodegradation property usually prior to delivery ability of the drug. However, major concerns associated with biodegradable polymeric nanoparticles are the acidic degradation by-products that can alter the drug activity and adversely interact with tissue. Most of CaPs are relatively insoluble in physiological environments, and this nature occurrence is one of the primary advantages over other synthetic drug delivery systems [14]. Except for the drugs, CaPs have also proven to be effective for non-viral intracellular gene delivery or transfection [15–17]. The use of CaP particle systems for protein delivery has also been studied using model proteins (e.g. bovine serum albumin) [18,19].
⁎
Up to now, CaPs have been used successfully in various drug delivery applications in the form of nanoscale (particulate systems) to microscale (coatings) to macroscale (cements and scaffolds) for local delivery, and in some cases for targeted delivery [13]. The recent researches are focusing on the better understanding of the microenvironment around the implant-host tissue interface, aiming to improve the osteogenic potential of bone substitutes in healthy bone sites and to provide a bone response in pathological ones [20]. With the purpose of improving the osteogenic potential of CaPs delivery, ion substitution may be a more appropriate way, for example strontium (Sr). Sr, which is demonstrated as a trace element in the human body, regulates bone remodeling effectively by stimulating bone formation and inhibiting bone resorption [21–23]. Numerous Sr substitution strategies have been developed for the efficient controlled release of bioactive strontium ions (Sr2 +) [24–28]. For bone regeneration, a Sr carrier with appropriate biodegradability and biocompatibility can exactly suit the demands of dynamic uptake and release of bone mineral ions, since almost all of Sr exists in the mineral metabolism region of bone tissue [29]. Among these Sr carriers, strontium hydrogen phosphate (SrHPO4, DSPA), as one of the main
Corresponding author. Correspondence to: J. Ye, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, PR China. E-mail addresses: [email protected] (T. Yu), [email protected] (J. Ye).
⁎⁎
http://dx.doi.org/10.1016/j.msec.2017.07.034 Received 13 June 2017; Received in revised form 5 July 2017; Accepted 19 July 2017 Available online 20 July 2017 0928-4931/ © 2017 Elsevier B.V. All rights reserved.
Materials Science & Engineering C 81 (2017) 8–12
H. Shi et al.
Fig. 1. XRD patterns, SEM images and EDS spectra of CaP (a–d) and SrP (e–h). EDS spectra were detected from the positions marked by red asterisks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
phosphatase (ALP) expressed by BMSCs. Cells were harvested and lysed by Triton X-100 (1 vol%). The cell lysates were quantified with a protein assay kit (Pierce BCA Protein Assay Kit, Thermo Scientific, USA). ALP activity was determined by measuring the absorbance of the paranitrophenol products using a para-nitrophenyl phosphate substrate (Sigma, USA). ALP staining was performed using BCIP/NBT substrate (SouthernBiotech, USA). Osteogenic differentiation mRNA expression of ALP, runt-related transcription factor 2 (Runx2), collagen 1 (Col1), osteocalcin (OCN) were detected by RT-PCR analysis using GAPDH as the housekeeping gene (Table S1). The total RNA in cells was isolated with HiPure Total RNA Micro Kit (Magen, China), subjected with iScript cDNA Synthesis Kit (Bio-Rad, USA), and then subjected to RTPCR analysis conducted with SYBR green assay (iQ™ SYBR Green Supermix, Bio-Rad, USA). Cellular tests were repeated four replicate samples and quantitative data were compared by a one-way analysis of variance (ANOVA) analysis at a significance level of P < 0.05.
compounds, has been recognized as an ion exchanger biomaterial for holding both HPO42 − and Sr2 + involved in the bone mineral metabolism [30]. SrHPO4 has been mainly claimed as an important catalyst, proton conductor and surface conditioner as well as for application in batteries, fuel cells, flame proofing and thermal cathodes. SrHPO4 is also usually used for the immobilization of heavy metal ions (e.g. Pb2 +) from the environmental sewage [31]. But in the biomedical fields, SrHPO4 is merely reported as a kind of Sr2 + additives in the CaPs biomaterials. SrHPO4 has been described with three types of modifications: α-SrHPO4 contains two independent Sr sites with eightfold coordination to form a distorted quadratic antiprism; γ-SrHPO4 includes two independent Sr sites coordinated by nine oxygen atoms; whereas little is known with concern to β-SrHPO4. In this work, β-SrHPO4 clusters with dandelion-like structure and function would be prepared as a novel drug delivery system. Biocompatibility evaluation including cellular adhesion, spreading, proliferation, osteogenic differentiation and mRNA expression would be employed to characterize its superior biological response using a bioactive CaP as the control. We believe that β-SrHPO4 clusters could be a suitable drug delivery system as bone substitute for the synergistic effects of bioactive Sr2 + release and unique dandelion-like structure.
3. Results and discussion OCP, which is proposed as an important bioactive precursor of biological apatite, has great potential to enhance new bone formation [33,34]. With the typical characteristics and structure of CaPs, its osteoconductive capacities were first demonstrated in granular form within the subperiosteal region of mouse calvaria, which demonstrated the rapid formation of new bone tissue more clearly than other CaPs (e.g. hydroxyapatite and Ca-deficient hydroxyapatite) [35]. OCP also has the potential to support the osteogenic differentiation of BMSCs [36,37]. In this work, bioactive OCP was employed as a control of CaPs for the biocompatibility evaluation of our SrHPO4 material. Phase component, morphology and structure of both powder samples are showed in Fig. 1. CaP and SrP were composed of exclusive OCP (Ca8H2(PO4)6·5H2O, PDF#26-1056) and β-SrHPO4 (PDF#12-0368), respectively. OCP crystals displayed typical plate-like appearance, while dandelion-like β-SrHPO4 cluster (Φ10–20 μm) consisted of numerous thin flaky crystals were observed. It was different from the general cube-shaped α-SrHPO4 crystals (PDF#70-1215, Fig. S1) which were structurally like DCPD (CaHPO4·2H2O) crystals. It was reported that the rapid cooling of the process temperature resulted in the formation of α-SrHPO4, whereas a continuous slow-heating process led to the appearance of β-SrHPO4 [38]. Here, carbamide could be another critical factor which could stabilize the newly formed β-SrHPO4 crystals despite our rapid cooling process. OCP crystals formed via the in-situ structural transition of rapidly precipitated plate-like DCPD crystals under the continuous hydrolysis of carbamide as reported in our previous work [32]. Similarly, the uniform β-SrHPO4 clusters could also epitaxially grow from the rapid formation of nuclei of strontium
2. Experimental procedure Powder samples were prepared by a homogeneous precipitation method as described in our previous work [32]. Briefly, white precipitation was obtained by vigorously stirring a solution containing 20 mM Ca(CH3COO)2/Sr(CH3COO)2, 15 mM NH4H2PO4 and 50 mM CO (NH2)2 at 90 °C for 2 h, followed by wash, centrifugation and air drying. Obtained samples were labelled as CaP and SrP, respectively. Phase component was determined by X-ray diffraction (XRD; X'Pert PRO, PANalytical, Netherlands). Morphology was observed by field emission scanning electron microscopy (FESEM; Nova NanoSEM 430, FEI, USA) with energy dispersive spectroscopy (EDS; Inca Energy, Oxford Instruments, UK). The in vitro biocompatibility was assessed in terms of biological response of bone mesenchymal stem cells (BMSCs; CRL-12424, ATCC, USA). Cells were seeded onto the culture plates (37 °C, 5%CO2), and powder samples were uniformly added after adhesion. Cellular morphology was observed by SEM after fixation and dehydration process. Cellular viability was observed with live/dead cell staining using an inverted fluorescence microscope (Eclipsc Ti-U, Nikon, Japan), and quantitatively assessed by CCK-8 assay (Dojindo, Japan) using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific, USA). Osteogenic differentiation was assessed by the amount of alkaline 9
Materials Science & Engineering C 81 (2017) 8–12
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Fig. 2. SEM images and live/dead staining fluorescent photographs of BMSCs co-cultured with CaP and SrP. Spread and aggregated cells were marked by red arrows and dotted line circles, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of cells was detected for β-SrHPO4 clusters than that of the control (Fig. 3a), which was in accordance with the fluorescence analysis above. ALP is the key marker of osteogenic differentiation of BMSCs, and its activity is one of the most important indicators employed for the evaluation of osteogenesis [40]. ALP expression of BMSCs on β-SrHPO4 clusters was at a significantly higher level according to the greater ALP activity of cells compared with the control (Fig. 3b). ALP staining observation had also provided evidences for the enhanced osteogenic effects of β-SrHPO4 clusters (Fig. 3g). Interestingly, numerous shining beads, which were attributed to β-SrHPO4 clusters, were clearly found concentrated in the dark stained areas of protein secretion. At the molecular level, the mRNA expressions of osteogenesis related genes (including ALP, Runx2, Col1 and OCN) of BMSCs further confirmed the remarkably greater osteogenic ability of β-SrHPO4 clusters than that of the control (Fig. 3c–f). The results indicated that the levels of osteogenic differentiation were specifically up-regulated by β-SrHPO4 clusters. In summary, compared with bioactive OCP crystals, β-SrHPO4 clusters showed better biocompatibility and excellent osteogenic activity, which could be suitably used as one of the promising bone substitutes in further applications. Herein, we proposed a brief scheme for the crystal nucleation and growth of synthetic dandelion-like β-SrHPO4 clusters as well as the potential application strategy as a novel drug carrier, as shown in Scheme 1. Rapid aggregation of Sr2 + and HPO42 − contributed to the formation of strontium phosphates nucleus in this reaction conditions. Flake and thin plate-like β-SrHPO4 clusters then gradually grew from the central nucleus to a radial dandelion-like microstructure. Due to the unique topological structure and bioactive Sr2 + release, β-SrHPO4
phosphate complexes accompanied with the uniform decomposition of carbamide [39]. Serval small plate-like β-SrHPO4 crystals had been observed in accordance with the developments of flaky crystals (Fig. 1g). Moreover, pH value of the reaction system continuously stayed at a low level caused by the consumption of HPO42 − and the release of H+, maintaining the stability of β-SrHPO4 crystals without further conversion to strontium hydroxyapatite (SAP, Sr10(PO4)6(OH)2). The results were indicated that pure β-SrHPO4 clusters with uniform sizes had been synthesized in the carbamide mediated homogeneous system. Adhesion, spreading morphology and viability of BMSCs co-cultured with CaP and SrP are displayed in Fig. 2. BMSCs attached and spread well on both samples. Importantly, these cells, which previously adhered on the plate, had crawled up the crystals, showing good cellular affinity. Moreover, cells revealed better adhesion ability onto β-SrHPO4 clusters accompanied with excellent fusion status and their fastened coupling. In contrast, the elongated spindle-shaped cells without fully spreading were observed for OCP crystals. As culture time prolonged, all the cells showed stable behaviors of division and growth, indicating that OCP crystals and β-SrHPO4 clusters had not inhibited the cellular growth ability. Compared with the control, β-SrHPO4 clusters significantly promoted the cellular viability. Specially, stronger fluorescence intensity of viable cells was clearly detected in the area where βSrHPO4 clusters gathered (marked by the red dotted line circles at day 5). Hence, β-SrHPO4 clusters had revealed notably enhanced cellular affinity, adhesion and viability than bioactive metastable OCP. Proliferation and osteogenic differentiation of BMSCs co-cultured with CaP and SrP are presented in Fig. 3. Greater proliferation capacity
Fig. 3. Proliferation (a), ALP activity (b), osteogenic differentiation gene expression (c–f) and ALP staining photographs (g) of BMSCs co-cultured with CaP and SrP. *SrP compared with CaP. ***P < 0.001.
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Scheme 1. Brief scheme for the crystal behaviors of synthetic dandelion-like β-SrHPO4 clusters (i) and the potential application strategy as a novel drug carrier (ii).
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clusters demonstrated excellent biological performances than OCP crystals. Moreover, a novel drug carrier will be developed with good drug delivery based on the gradual disintegration of biodegradable crystal cluster units in vivo, which is like the dandelion with its seeds gone with the wind. 4. Conclusions Dandelion-like β-SrHPO4 clusters (Φ10–20 μm) constituted by flaky crystals were successfully prepared through a homogeneous precipitation method under the hydrolysis of carbamide. Compared with the control, the highly osteoconductive and biodegradable plate-like OCP crystals, β-SrHPO4 clusters exhibited significantly better affinity, proliferation and osteogenic differentiation of BMSCs. It will provide a potential alternative drug delivery for tissue repair, and broaden the further biomedical applications of this energy material. Further investigations of β-SrHPO4 clusters in drug delivery system and bone regeneration in vivo will be made in our follow-up researches. Acknowledgements This research was supported by the National Key Research and Development Program of China (2016YFB0700803), the Science and Technology Program of Guangzhou City of China (201508020017), the Fundamental Research Funds for the Central Universities, the China Postdoctoral Science Foundation (2016M602601) and the National Natural Science Foundation of China (31670969). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2017.07.034. References [1] R. Lakshminarayanan, E.O. Chi-Jin, X.J. Loh, R.M. Kini, S. Valiyaveettil, Purification and characterization of a vaterite-inducing peptide, pelovaterin, from the eggshells of Pelodiscus sinensis (Chinese soft-shelled turtle), Biomacromolecules 6 (2005) 1429–1437. [2] R. Lakshminarayanan, X.J. Loh, S. Gayathri, Y. Banerjee, R.M. Kini, S. Valiyaveettil, Formation of transient amorphous calcium carbonate precursor in quail eggshell mineralization: an in vitro study, Biomacromolecules 7 (2006) 3202–3209. [3] E. Ye, X.J. Loh, Polymeric hydrogels and nanoparticles: a merging and emerging field, Aust. J. Chem. 66 (2013) 997–1007. [4] Q.Q. Dou, C.P. Teng, E. Ye, X.J. Loh, Effective near-infrared photodynamic therapy assisted by upconversion nanoparticles conjugated with photosensitizers, Int. J. Nanomedicine 10 (2015) 419–432. [5] C. Dhand, N. Dwivedi, X.J. Loh, A.N.J. Ying, N.K. Verma, R.W. Beuerman, R. Lakshminarayanan, S. Ramakrishna, Methods and strategies for the synthesis of diverse nanoparticles and their applications: a comprehensive overview, RSC Adv. 5
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12
Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Short communication
Biocompatible β-SrHPO4 clusters with dandelion-like structure as an alternative drug carrier
MARK
Haishan Shia,b,c, Tingting Wua,b,c, Jing Zhangb,c, Xiaoling Yeb,c, Shenghui Zenga, Xu Liua, Tao Yua,⁎, Jiandong Yeb,c,⁎⁎, Changren Zhoua a b c
College of Chemistry and Materials, Jinan University, Guangzhou 510632, PR China School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, PR China National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Strontium hydrogen phosphate Calcium phosphate Bone mesenchymal stem cells
Recent researches about calcium phosphate (CaP) biomaterials used as drug delivery systems are focusing on the better understanding of the microenvironment around the implant-host tissue interface, with the aim to provide a bone response in pathological ones. Towards the improvement of the osteogenic potential of CaP drug carriers, dandelion-like β-SrHPO4 clusters (Φ10–20 μm) has been prepared by a homogeneous precipitation method under the hydrolysis of carbamide. Adhesion, spreading, proliferation, osteogenic differentiation and mRNA expression of bone mesenchymal stem cells (BMSCs) mediated by β-SrHPO4 clusters were investigated. Highly osteoconductive and biodegradable octacalcium phosphate with similar structure was employed as the control. By contrast, β-SrHPO4 clusters exhibited remarkably better affinity, enhanced proliferation and osteogenic differentiation of BMSCs, providing a promising alternative bioactive bone substitute and drug carrier for tissue repair. With the unique dandelion-like microstructure, we believe that our as-prepared material will open up new avenues for applicability of CaP drug delivery systems in the near future.
1. Introduction Recently, hybrid materials and inorganic materials are widely recognized as upcoming materials for delivery, therapy, diagnosis, imaging, sensors, etc. in optical, electrical, biomedical, medical, clinical and other applications [1–12]. With the compositional similarities to bone minerals and excellent biocompatibility, calcium phosphates (CaPs) are widely used in bone regeneration. CaPs have become the research focus in simultaneous use as bone substitute and drug delivery vehicle [13]. As drug carriers, the biodegradation property usually prior to delivery ability of the drug. However, major concerns associated with biodegradable polymeric nanoparticles are the acidic degradation by-products that can alter the drug activity and adversely interact with tissue. Most of CaPs are relatively insoluble in physiological environments, and this nature occurrence is one of the primary advantages over other synthetic drug delivery systems [14]. Except for the drugs, CaPs have also proven to be effective for non-viral intracellular gene delivery or transfection [15–17]. The use of CaP particle systems for protein delivery has also been studied using model proteins (e.g. bovine serum albumin) [18,19].
⁎
Up to now, CaPs have been used successfully in various drug delivery applications in the form of nanoscale (particulate systems) to microscale (coatings) to macroscale (cements and scaffolds) for local delivery, and in some cases for targeted delivery [13]. The recent researches are focusing on the better understanding of the microenvironment around the implant-host tissue interface, aiming to improve the osteogenic potential of bone substitutes in healthy bone sites and to provide a bone response in pathological ones [20]. With the purpose of improving the osteogenic potential of CaPs delivery, ion substitution may be a more appropriate way, for example strontium (Sr). Sr, which is demonstrated as a trace element in the human body, regulates bone remodeling effectively by stimulating bone formation and inhibiting bone resorption [21–23]. Numerous Sr substitution strategies have been developed for the efficient controlled release of bioactive strontium ions (Sr2 +) [24–28]. For bone regeneration, a Sr carrier with appropriate biodegradability and biocompatibility can exactly suit the demands of dynamic uptake and release of bone mineral ions, since almost all of Sr exists in the mineral metabolism region of bone tissue [29]. Among these Sr carriers, strontium hydrogen phosphate (SrHPO4, DSPA), as one of the main
Corresponding author. Correspondence to: J. Ye, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, PR China. E-mail addresses: [email protected] (T. Yu), [email protected] (J. Ye).
⁎⁎
http://dx.doi.org/10.1016/j.msec.2017.07.034 Received 13 June 2017; Received in revised form 5 July 2017; Accepted 19 July 2017 Available online 20 July 2017 0928-4931/ © 2017 Elsevier B.V. All rights reserved.
Materials Science & Engineering C 81 (2017) 8–12
H. Shi et al.
Fig. 1. XRD patterns, SEM images and EDS spectra of CaP (a–d) and SrP (e–h). EDS spectra were detected from the positions marked by red asterisks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
phosphatase (ALP) expressed by BMSCs. Cells were harvested and lysed by Triton X-100 (1 vol%). The cell lysates were quantified with a protein assay kit (Pierce BCA Protein Assay Kit, Thermo Scientific, USA). ALP activity was determined by measuring the absorbance of the paranitrophenol products using a para-nitrophenyl phosphate substrate (Sigma, USA). ALP staining was performed using BCIP/NBT substrate (SouthernBiotech, USA). Osteogenic differentiation mRNA expression of ALP, runt-related transcription factor 2 (Runx2), collagen 1 (Col1), osteocalcin (OCN) were detected by RT-PCR analysis using GAPDH as the housekeeping gene (Table S1). The total RNA in cells was isolated with HiPure Total RNA Micro Kit (Magen, China), subjected with iScript cDNA Synthesis Kit (Bio-Rad, USA), and then subjected to RTPCR analysis conducted with SYBR green assay (iQ™ SYBR Green Supermix, Bio-Rad, USA). Cellular tests were repeated four replicate samples and quantitative data were compared by a one-way analysis of variance (ANOVA) analysis at a significance level of P < 0.05.
compounds, has been recognized as an ion exchanger biomaterial for holding both HPO42 − and Sr2 + involved in the bone mineral metabolism [30]. SrHPO4 has been mainly claimed as an important catalyst, proton conductor and surface conditioner as well as for application in batteries, fuel cells, flame proofing and thermal cathodes. SrHPO4 is also usually used for the immobilization of heavy metal ions (e.g. Pb2 +) from the environmental sewage [31]. But in the biomedical fields, SrHPO4 is merely reported as a kind of Sr2 + additives in the CaPs biomaterials. SrHPO4 has been described with three types of modifications: α-SrHPO4 contains two independent Sr sites with eightfold coordination to form a distorted quadratic antiprism; γ-SrHPO4 includes two independent Sr sites coordinated by nine oxygen atoms; whereas little is known with concern to β-SrHPO4. In this work, β-SrHPO4 clusters with dandelion-like structure and function would be prepared as a novel drug delivery system. Biocompatibility evaluation including cellular adhesion, spreading, proliferation, osteogenic differentiation and mRNA expression would be employed to characterize its superior biological response using a bioactive CaP as the control. We believe that β-SrHPO4 clusters could be a suitable drug delivery system as bone substitute for the synergistic effects of bioactive Sr2 + release and unique dandelion-like structure.
3. Results and discussion OCP, which is proposed as an important bioactive precursor of biological apatite, has great potential to enhance new bone formation [33,34]. With the typical characteristics and structure of CaPs, its osteoconductive capacities were first demonstrated in granular form within the subperiosteal region of mouse calvaria, which demonstrated the rapid formation of new bone tissue more clearly than other CaPs (e.g. hydroxyapatite and Ca-deficient hydroxyapatite) [35]. OCP also has the potential to support the osteogenic differentiation of BMSCs [36,37]. In this work, bioactive OCP was employed as a control of CaPs for the biocompatibility evaluation of our SrHPO4 material. Phase component, morphology and structure of both powder samples are showed in Fig. 1. CaP and SrP were composed of exclusive OCP (Ca8H2(PO4)6·5H2O, PDF#26-1056) and β-SrHPO4 (PDF#12-0368), respectively. OCP crystals displayed typical plate-like appearance, while dandelion-like β-SrHPO4 cluster (Φ10–20 μm) consisted of numerous thin flaky crystals were observed. It was different from the general cube-shaped α-SrHPO4 crystals (PDF#70-1215, Fig. S1) which were structurally like DCPD (CaHPO4·2H2O) crystals. It was reported that the rapid cooling of the process temperature resulted in the formation of α-SrHPO4, whereas a continuous slow-heating process led to the appearance of β-SrHPO4 [38]. Here, carbamide could be another critical factor which could stabilize the newly formed β-SrHPO4 crystals despite our rapid cooling process. OCP crystals formed via the in-situ structural transition of rapidly precipitated plate-like DCPD crystals under the continuous hydrolysis of carbamide as reported in our previous work [32]. Similarly, the uniform β-SrHPO4 clusters could also epitaxially grow from the rapid formation of nuclei of strontium
2. Experimental procedure Powder samples were prepared by a homogeneous precipitation method as described in our previous work [32]. Briefly, white precipitation was obtained by vigorously stirring a solution containing 20 mM Ca(CH3COO)2/Sr(CH3COO)2, 15 mM NH4H2PO4 and 50 mM CO (NH2)2 at 90 °C for 2 h, followed by wash, centrifugation and air drying. Obtained samples were labelled as CaP and SrP, respectively. Phase component was determined by X-ray diffraction (XRD; X'Pert PRO, PANalytical, Netherlands). Morphology was observed by field emission scanning electron microscopy (FESEM; Nova NanoSEM 430, FEI, USA) with energy dispersive spectroscopy (EDS; Inca Energy, Oxford Instruments, UK). The in vitro biocompatibility was assessed in terms of biological response of bone mesenchymal stem cells (BMSCs; CRL-12424, ATCC, USA). Cells were seeded onto the culture plates (37 °C, 5%CO2), and powder samples were uniformly added after adhesion. Cellular morphology was observed by SEM after fixation and dehydration process. Cellular viability was observed with live/dead cell staining using an inverted fluorescence microscope (Eclipsc Ti-U, Nikon, Japan), and quantitatively assessed by CCK-8 assay (Dojindo, Japan) using a microplate reader (Varioskan Flash Multimode Reader, Thermo Scientific, USA). Osteogenic differentiation was assessed by the amount of alkaline 9
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Fig. 2. SEM images and live/dead staining fluorescent photographs of BMSCs co-cultured with CaP and SrP. Spread and aggregated cells were marked by red arrows and dotted line circles, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of cells was detected for β-SrHPO4 clusters than that of the control (Fig. 3a), which was in accordance with the fluorescence analysis above. ALP is the key marker of osteogenic differentiation of BMSCs, and its activity is one of the most important indicators employed for the evaluation of osteogenesis [40]. ALP expression of BMSCs on β-SrHPO4 clusters was at a significantly higher level according to the greater ALP activity of cells compared with the control (Fig. 3b). ALP staining observation had also provided evidences for the enhanced osteogenic effects of β-SrHPO4 clusters (Fig. 3g). Interestingly, numerous shining beads, which were attributed to β-SrHPO4 clusters, were clearly found concentrated in the dark stained areas of protein secretion. At the molecular level, the mRNA expressions of osteogenesis related genes (including ALP, Runx2, Col1 and OCN) of BMSCs further confirmed the remarkably greater osteogenic ability of β-SrHPO4 clusters than that of the control (Fig. 3c–f). The results indicated that the levels of osteogenic differentiation were specifically up-regulated by β-SrHPO4 clusters. In summary, compared with bioactive OCP crystals, β-SrHPO4 clusters showed better biocompatibility and excellent osteogenic activity, which could be suitably used as one of the promising bone substitutes in further applications. Herein, we proposed a brief scheme for the crystal nucleation and growth of synthetic dandelion-like β-SrHPO4 clusters as well as the potential application strategy as a novel drug carrier, as shown in Scheme 1. Rapid aggregation of Sr2 + and HPO42 − contributed to the formation of strontium phosphates nucleus in this reaction conditions. Flake and thin plate-like β-SrHPO4 clusters then gradually grew from the central nucleus to a radial dandelion-like microstructure. Due to the unique topological structure and bioactive Sr2 + release, β-SrHPO4
phosphate complexes accompanied with the uniform decomposition of carbamide [39]. Serval small plate-like β-SrHPO4 crystals had been observed in accordance with the developments of flaky crystals (Fig. 1g). Moreover, pH value of the reaction system continuously stayed at a low level caused by the consumption of HPO42 − and the release of H+, maintaining the stability of β-SrHPO4 crystals without further conversion to strontium hydroxyapatite (SAP, Sr10(PO4)6(OH)2). The results were indicated that pure β-SrHPO4 clusters with uniform sizes had been synthesized in the carbamide mediated homogeneous system. Adhesion, spreading morphology and viability of BMSCs co-cultured with CaP and SrP are displayed in Fig. 2. BMSCs attached and spread well on both samples. Importantly, these cells, which previously adhered on the plate, had crawled up the crystals, showing good cellular affinity. Moreover, cells revealed better adhesion ability onto β-SrHPO4 clusters accompanied with excellent fusion status and their fastened coupling. In contrast, the elongated spindle-shaped cells without fully spreading were observed for OCP crystals. As culture time prolonged, all the cells showed stable behaviors of division and growth, indicating that OCP crystals and β-SrHPO4 clusters had not inhibited the cellular growth ability. Compared with the control, β-SrHPO4 clusters significantly promoted the cellular viability. Specially, stronger fluorescence intensity of viable cells was clearly detected in the area where βSrHPO4 clusters gathered (marked by the red dotted line circles at day 5). Hence, β-SrHPO4 clusters had revealed notably enhanced cellular affinity, adhesion and viability than bioactive metastable OCP. Proliferation and osteogenic differentiation of BMSCs co-cultured with CaP and SrP are presented in Fig. 3. Greater proliferation capacity
Fig. 3. Proliferation (a), ALP activity (b), osteogenic differentiation gene expression (c–f) and ALP staining photographs (g) of BMSCs co-cultured with CaP and SrP. *SrP compared with CaP. ***P < 0.001.
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Scheme 1. Brief scheme for the crystal behaviors of synthetic dandelion-like β-SrHPO4 clusters (i) and the potential application strategy as a novel drug carrier (ii).
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clusters demonstrated excellent biological performances than OCP crystals. Moreover, a novel drug carrier will be developed with good drug delivery based on the gradual disintegration of biodegradable crystal cluster units in vivo, which is like the dandelion with its seeds gone with the wind. 4. Conclusions Dandelion-like β-SrHPO4 clusters (Φ10–20 μm) constituted by flaky crystals were successfully prepared through a homogeneous precipitation method under the hydrolysis of carbamide. Compared with the control, the highly osteoconductive and biodegradable plate-like OCP crystals, β-SrHPO4 clusters exhibited significantly better affinity, proliferation and osteogenic differentiation of BMSCs. It will provide a potential alternative drug delivery for tissue repair, and broaden the further biomedical applications of this energy material. Further investigations of β-SrHPO4 clusters in drug delivery system and bone regeneration in vivo will be made in our follow-up researches. Acknowledgements This research was supported by the National Key Research and Development Program of China (2016YFB0700803), the Science and Technology Program of Guangzhou City of China (201508020017), the Fundamental Research Funds for the Central Universities, the China Postdoctoral Science Foundation (2016M602601) and the National Natural Science Foundation of China (31670969). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2017.07.034. References [1] R. Lakshminarayanan, E.O. Chi-Jin, X.J. Loh, R.M. Kini, S. Valiyaveettil, Purification and characterization of a vaterite-inducing peptide, pelovaterin, from the eggshells of Pelodiscus sinensis (Chinese soft-shelled turtle), Biomacromolecules 6 (2005) 1429–1437. [2] R. Lakshminarayanan, X.J. Loh, S. Gayathri, Y. Banerjee, R.M. Kini, S. Valiyaveettil, Formation of transient amorphous calcium carbonate precursor in quail eggshell mineralization: an in vitro study, Biomacromolecules 7 (2006) 3202–3209. [3] E. Ye, X.J. Loh, Polymeric hydrogels and nanoparticles: a merging and emerging field, Aust. J. Chem. 66 (2013) 997–1007. [4] Q.Q. Dou, C.P. Teng, E. Ye, X.J. Loh, Effective near-infrared photodynamic therapy assisted by upconversion nanoparticles conjugated with photosensitizers, Int. J. Nanomedicine 10 (2015) 419–432. [5] C. Dhand, N. Dwivedi, X.J. Loh, A.N.J. Ying, N.K. Verma, R.W. Beuerman, R. Lakshminarayanan, S. Ramakrishna, Methods and strategies for the synthesis of diverse nanoparticles and their applications: a comprehensive overview, RSC Adv. 5
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