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Doping strontium in tricalcium phosphate microspheres using yeast-based biotemplate
Materials Chemistry and Physics 147 (2014) 540e544
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Doping strontium in tricalcium phosphate microspheres using yeast-based biotemplate Miaojun Huang a, b, 1, Tianjie Li a, b, 1, Naru Zhao a, b, Yongchang Yao a, Hui Yang a, Chang Du a, b, *, Yingjun Wang a, b, * a b
School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Yeast cells were used as biotemplate cores to prepare b-TCP microspheres. The approach to synthesis b-TCP microspheres was inspired by biomineralization. Sr2þ partially occupied Ca2þ sites in b-TCP crystal lattice. The amount of doped Sr2þ can be controlled with the original Sr2þ concentration. The Sr-doped b-TCP microspheres had an excellent biocompatibility.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 January 2014 Received in revised form 15 April 2014 Accepted 8 May 2014 Available online 10 June 2014
Tricalcium phosphate (TCP) microspheres carrying strontium were fabricated using yeast cells as a biotemplate and growing materials through co-precipitation around the biotemplate core. In order to dope Sr in the TCP, the biotemplate was allowed to pre-adsorb Sr2þ and Ca2þ before materials deposition. It was found b-TCP with Sr2þ partially occupying Ca2þ site was formed with high purity. The amount of Sr doped in b-TCP linearly increased with the original Sr2þ concentration adsorbed on the biotemplate. The cell toxicity tests using human marrow stromal cells (hMSCs) showed that Sr-doped TCP microspheres have an excellent biocompatibility. The product can serve as a potential drug release system for the bone regeneration field. © 2014 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Microstructure Organic compounds Precipitation
1. Introduction Calcium phosphate has been widely considered as one of the most important inorganic biomaterials due to the similarities in
* Corresponding authors. National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China. Tel.: þ86 20 87112160; fax: þ86 20 22236088. E-mail addresses: [email protected] (C. Du), [email protected] (Y. Wang). 1 M. Huang and T. Li are co-first authors, and they made an equal contribution to this work. http://dx.doi.org/10.1016/j.matchemphys.2014.05.028 0254-0584/© 2014 Elsevier B.V. All rights reserved.
composition and structure to natural bone and tooth minerals [1e5]. TCP is one of typical calcium phosphates, which has proved to show excellent biodegradability and biocompatibility [6e10]. In recent years, the biomimetic topic of the ionic substitution in TCP has been pursued [11e17] because the main inorganic minerals in biological bone components contain various elements. The effective role of these trace elements in biomaterials is well-known. They not only alter the physicochemical properties of calcium phosphate, but also have their own significances in the biological response. For instance, strontium as one of bone-seeking elements is distributed in bone, and plays an important biological role in
M. Huang et al. / Materials Chemistry and Physics 147 (2014) 540e544
human bone metabolic turnover [18,19]. It is generally accepted that Sr2þ can replace Ca2þ in the crystal lattice within a large concentration range owing to the chemical similarity. Its substitution results in the crystal destabilization and then the increasing solubility [20e22]. Furthermore, the low dose of Sr substitution has the beneficial influence on the decrease of bone resorption and the enhancement of bone formation by inhibiting osteoclast-resorbing function and stimulating osteoblast activity and differentiation [11,23,24]. Therefore, this work is focused on the doping of Sr in TCP to form Sr-TCP using a new biotemplating approach. Various synthetic methods for Sr-TCP powders have been intensively studied, such as the solid state calcination, coprecipitation, solegel process, and hydrothermal method [20,25e27]. There are commonly two ways, the solid-phase and liquid phase method. In the former method, the solid-phase Sr salts incorporate with calcium and phosphate salts to form Sr-TCP at a high sintering temperature [25]. However, it is quite difficult to completely mix the raw materials, and a subsequent milling process is necessary. Thus the product morphology and the Sr distribution have a poor controllability. By a liquid phase method, Sr2þ is incorporated into the amorphous calcium phosphate structure during the precipitation process, and then doped into the crystal lattice after calcinations, which is confirmed as a more effective way to obtain Sr-TCP particles. However, most traditional liquid phase method can only prepare the sheet/rod-like powders without any additives. The effective control over the composition, structure and morphology is still a challenge. In terms of the increasing scientific interest towards the biomimetic synthesis and the recognition for microspheres [28e32], we, along with others, have developed a biotemplate method which proves to be feasible in the fabrication of functional biomaterials [33e35]. Moreover our previous works have successfully prepared a calcium phosphate microcapsule via a biomimetic mineralization process using yeast cells as a template core [34]. In this work, we aim at developing an effective way to synthesize porous Sr-TCP microspheres via the yeast-based biotemplate method. The Sr dose in the microspheres can be effectively controlled. Moreover, the biocompatibility of Sr-TCP was evaluated. The final product was proposed to have potential applications in bone medical engineering.
2. Materials and methods 2.1. Synthesis Yeast cells (Saccharomyces cerevisiae) were isolated and purified from the ANGEL's instant dry yeast by the spread plate method and the streak plate method. They were cultured in YPD medium (1% yeast extract, 2% peptone, and 2% glucose), incubated at 28 C with shaking at 180 rpm. The synthesis of Sr-TCP microspheres was described in the reference [34]. Yeast cells were alternately immersed into Poly(diallyldimethylammonium chloride) (PDADMAC) and Poly(acrylic acid) (PAA) solution via the layer-by-layer treatment (LBL) and formed yeast/PDADMAC/PAA composite biotemplates. The biotemplates were mixed into calcium chloride (CaCl2) and strontium chloride (SrCl2) solution with different Sr/ (Sr þ Ca) molar ratios, stirring for 12 h. The biotemplates absorbed Sr2þ and Ca2þ were added to the diammonium phosphate ((NH4)2HPO4) solution, stirring for 12 h under ambient temperature. The pH value was regulated about 10. After centrifugation and washed, the product was dried and then calcined at 800 C. The corresponding products with different Sr/(Sr þ Ca) molar ratios (0, 1, 5, 10 and 15%) were denoted as Sr0, Sr1, Sr5, Sr10 and Sr15, respectively.
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2.2. Cell cytotoxic experiment The initial cell of the 3rd generation of human marrow stromal cells (hMSCs) was donated by the First Affiliated Hospital of Sun Yat-sen University in Guangzhou as described in the reference [36]. Cells were exposed to Sr-TCP powders via the following way [37]. Sr-TCP powders were autoclaved and stored in a sealed tube. The sample was re-suspended with the low sugar complete medium using ultrasound to ensure a predetermined final concentration (200 mg ml1). The cell suspension with the 7th generation of hMSCs was added into the suspension of Sr-TCP powder, using a cell counter counts to reach a specified concentration range (4 103 cells/hole). The mixture was feed in 96-well plates and placed in incubator (37 C, 5% CO2). The Cell Counting Kit-8 (CCK-8) was used to test the cell proliferation/toxicity experiment. Tetrazolium salt WST®-8 under the effect of 1-Methoxy PMS electron carrier is reduced to a watersoluble yellow formazan dye. The toxicity was estimated with measuring the absorbance of the solution. The absorbance value at 450 nm represents the number of living cells. The samples without powders and the one with a 0.65% phenol solution were regarded as the negative control and positive control, respectively. All samples were assayed at the time points (4 h, 1 day, 3 day and 7 day). 2.3. Characterization X-ray diffraction (XRD) patterns of samples were obtained on a Panalytical X'Pert PRO (PANalytical, Netherlands) using Cu Ka (l ¼ 0.15418 nm) radiation. The morphologies of the products were observed by the scanning electron microscopy (SEM, Hitachi H-800). The elements qualitative and semi-quantitative analysis of the sample was tested using the electron spectrometer (EDS, EPMA-1600, Shimadzu Corporation). The CCK-8 assay was detected using the multifunctional microplate reader (Thermo3001, U.S. Thermo Electro Corporation). 3. Result and discussion Fig. 1 shows morphologies of yeast cells before and after mineralization, as well as porous Sr-TCP microspheres calcined at 800 C. The yeast-based biotemplates method was proved to be an effective way to prepared calcium phosphate microspheres. Yeast cells were shrivelled after the freeze dry treatment with a soft surface (Fig. 1A1eA3). After the mineralization, particles appeared to be plump and presented a complete spherical structure with a diameter about 5 mm (Fig. 1B1eB2). Numbers of cluster-like protuberances appeared on the rough surface (Fig. 1B3). The microsphere structure remained after calcination at 800 C (Fig. 1C1eC3). Crystal grains were neck-like connected and made up of the porous shell. The fine nano-scale pores on the surface of the microsphere have an average diameter of 120 ± 14 nm based on the measurement from SEM photos. In addition, the microsphere had a hollow structure resulting from the biotemplate removing under heat treatment. From some broken microspheres, the porosity was estimated with a simple spherical model. The diameter of the microsphere and the thickness of shell were measured on SEM photos. The average porosity of microsphere was about 70% ± 4% (v/v) (n ¼ 10), which was calculated by using the equation
P¼
10 1 X Di 2di 3 100% 10 i¼1 Di
(1)
where Di is the spherical diameter of the ith microsphere, di is the thickness of the ith shell, and P is the average porosity of
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M. Huang et al. / Materials Chemistry and Physics 147 (2014) 540e544
Fig. 1. Morphologies of yeast cells before and after mineralization as well as Sr-TCP microspheres calcined at 800 C. A1-A3 represents the different magnifications of dry yeast cells via the freeze drying treatment. B1eB3 represents the different magnifications of Sr-TCP microspheres prepared by using the yeast-based biotemplates method before calcined. C1eC3 represents the different magnifications of Sr-TCP particles calcined at 800 C.
microsphere. Compared to the TCP microcapsule without Sr2þ addition (Seen in Support Information), the protuberances before calcination and smaller nanocrystals after calcination on the microsphere surface may result from the Sr2þ incorporation in TCP. The Sr2þ substitution can affect the TCP crystal lattice and lead to the morphological change [21]. Fig. 2 shows the XRD pattern of the products obtained with different original Sr/(Sr þ Ca) ratios. According to the JCPDS database 00-009-0169, the b-TCP phase (Ca3(PO4)2, whitlockite) was present in all samples (Fig. 2B). However, with the Sr/(Sr þ Ca) ratios increasing from 0 to 15 at%, XRD patterns gradually shifted to the lower angle, and the diffraction intensity of main characteristic peaks at 31.0 , 27.8 and 34.4 weakened (Fig. 2A). These results suggested that Sr2þ partly replaced Ca2þ in TCP crystal lattice
[38,39]. Since the Sr2þ radius (1.13 Å) is larger than Ca2þ (0.96 Å), the difference of ionic radius caused part of the lattice distortion. Lattice parameters (a and c-axis) as well as the unit cell volume linearly increase with Sr-addition [39]. The diffraction angle and the characteristic peak intensity were thus slightly altered. The Sr substitution can hinder the normal grain growth, which further changed the surface morphology of TCP microspheres (shown in Fig. 1). Besides, the shift degree directly related to the Sr content in particles which was tested in Fig. 3. Fig. 3 shows the EDS analysis of Sr-TCP powders with different original Sr/(Sr þ Ca) ratios. Elements of Ca, P, O and C were clearly shown (Fig. 3B). Ca, P and O elements belonged to TCP, while C elements may be derived from the CO2 in surrounding environment or a small amount of CO2 3 substitution during calcinations [34].
Fig. 2. XRD patterns of Sr-TCP powders calcined at 800 C. Samples denoted as Sr0, Sr1, Sr5, Sr10 and Sr15 represents the product derived from different original ratios of Sr/ (Sr þ Ca) at 0, 1, 5, 10 and 15 at%, respectively. A was the magnified version of the shadowed area in B.
M. Huang et al. / Materials Chemistry and Physics 147 (2014) 540e544
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Fig. 3. EDS analysis of Sr-TCP products calcined at 800 C with different original Sr doses represented by the original ratios of Sr/(Sr þ Ca). A represents the atomic percentage of Sr to Sr þ Ca in Sr-TCP calculated from EDS spectra shown in B. B represents the EDS analysis for all elements of Sr-TCP.
Particularly, the Sr characteristic peak emerged with Sr-addition. Its intensity increased linearly with the original Sr/(Sr þ Ca) ratios gradually increasing from 0 to 15 at% (Fig. 3A). The atomic percentage of Sr to Sr þ Ca in Sr-TCP showed a linear incorporation of Sr through the substitution of Ca in TCP lattice. The increasing Sr content may lead to the shift of diffraction peaks in XRD patterns. The result showed that the Sr content in TCP microspheres can be effectively controlled via the regulation of original Sr/(Sr þ Ca) ratios. Moreover, Sr2þ released slowly in a long time when Sr-TCP powders immerged in the phosphate buffered saline (seen in Support information). Fig. 4 shows morphologies of hMSCs cultured with Sr-TCP powders at 7 day. The fusiform living cells evenly overspread on the plate bottom without adding powders (Fig. 4A). However, the
cells' distribution was disturbed after adding Sr-TCP powders (Fig. 4B). Cells were inclined to be an aggregation. From light field optical images, particles were observed to be a banding-like distribution (Fig. 4C), which may result from the particle adhesion onto the cell surface. Block particle aggregations were present (Fig. 4D). We believe that the block can provide the better field for cell adhesion and conduct the cell growth to the three-dimensional space. Thus cells densely adhered and climbed around block aggregations, which may result in the appearance of cells' aggregations in Fig. 4B. The result indicates that the Sr-TCP powders have an excellent biocompatibility with hMSCs. Further demonstration about Sr-TCP biocompatibility was shown in Fig. 5. The CCK-8 assay result indicated that OD450nm values of all experimental samples significantly increased with the
Fig. 4. Morphologies of hMSCs cultured with Sr-TCP powders at day 7. A represents the fluorescent image from the hMSCs sample without adding Sr-TCP powders. B represents the fluorescent image from the hMSCs sample after interaction with Sr-TCP powders. Living cells show green emission. C and D represent the different areas of the bright field optical image taken from the hMSCs sample after interaction with Sr-TCP powders. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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M. Huang et al. / Materials Chemistry and Physics 147 (2014) 540e544
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.matchemphys.2014.05.028.
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
[10] Fig. 5. The CCK-8 analysis from controls and experimental hMSCs samples after cocultured with Sr-TCP powders for 4 h, 1 d, 3 d and 7 d. Positive and negative control were obtained from the sample with 0.65wt% phenol solution and the blank plate. (&, # and Ф indicated statistical significance when the experiment group was compared with the positive control, negative control, and the sample Sr0 without adding Sr2þ, respectively, p < 0.05).
prolonged incubation time. Compared to the positive control, all SrTCP powders showed statistically significant (p < 0.05) promotion on the hMSCs proliferation. Except the sample Sr1, OD450nm values of the rest experimental samples indicated statistical significance compared with the negative control and Sr0 (p < 0.05) after 7 days' culture. The result indicated the proliferation rate of hMSCs cocultured with Sr-TCP powders is always higher than that with pure TCP, even higher than the negative control in blank plate. Therefore, Sr-TCP powders prepared via yeast-based biotemplate method were confirmed to be beneficial for the hMSCs proliferation. 4. Conclusions
[11] [12]
[13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
A yeast-based biotemplating method was introduced for the effective synthesis of Sr-TCP microspheres. The porous Sr-TCP microsphere with a diameter of approximately 5 mm was obtained by allowing the biotemplate to pre-adsorb a mixture solution of Sr2þ and Ca2þ with different original Sr/(Sr þ Ca) molar ratios. The pre-adsorption enables the doping of Sr2þ in TCP crystal lattice by partially substituting Ca2þ. The amount of Sr doped in TCP can be effectively controlled. The Sr-TCP powder exhibited an excellent biocompatibility with hMSCs. We propose the final product can be used as a drug carrier and applied in the medical field for the bone regeneration. Acknowledgements This work was supported by National Basic Research Program of China (2012CB619100), National Nature Science Foundation of China (50732003, U0834003, 51072056), and Program for Changjiang Scholars and Innovative Research Team in University (IRT 0919).
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Doping strontium in tricalcium phosphate microspheres using yeast-based biotemplate Miaojun Huang a, b, 1, Tianjie Li a, b, 1, Naru Zhao a, b, Yongchang Yao a, Hui Yang a, Chang Du a, b, *, Yingjun Wang a, b, * a b
School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Yeast cells were used as biotemplate cores to prepare b-TCP microspheres. The approach to synthesis b-TCP microspheres was inspired by biomineralization. Sr2þ partially occupied Ca2þ sites in b-TCP crystal lattice. The amount of doped Sr2þ can be controlled with the original Sr2þ concentration. The Sr-doped b-TCP microspheres had an excellent biocompatibility.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 January 2014 Received in revised form 15 April 2014 Accepted 8 May 2014 Available online 10 June 2014
Tricalcium phosphate (TCP) microspheres carrying strontium were fabricated using yeast cells as a biotemplate and growing materials through co-precipitation around the biotemplate core. In order to dope Sr in the TCP, the biotemplate was allowed to pre-adsorb Sr2þ and Ca2þ before materials deposition. It was found b-TCP with Sr2þ partially occupying Ca2þ site was formed with high purity. The amount of Sr doped in b-TCP linearly increased with the original Sr2þ concentration adsorbed on the biotemplate. The cell toxicity tests using human marrow stromal cells (hMSCs) showed that Sr-doped TCP microspheres have an excellent biocompatibility. The product can serve as a potential drug release system for the bone regeneration field. © 2014 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Microstructure Organic compounds Precipitation
1. Introduction Calcium phosphate has been widely considered as one of the most important inorganic biomaterials due to the similarities in
* Corresponding authors. National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China. Tel.: þ86 20 87112160; fax: þ86 20 22236088. E-mail addresses: [email protected] (C. Du), [email protected] (Y. Wang). 1 M. Huang and T. Li are co-first authors, and they made an equal contribution to this work. http://dx.doi.org/10.1016/j.matchemphys.2014.05.028 0254-0584/© 2014 Elsevier B.V. All rights reserved.
composition and structure to natural bone and tooth minerals [1e5]. TCP is one of typical calcium phosphates, which has proved to show excellent biodegradability and biocompatibility [6e10]. In recent years, the biomimetic topic of the ionic substitution in TCP has been pursued [11e17] because the main inorganic minerals in biological bone components contain various elements. The effective role of these trace elements in biomaterials is well-known. They not only alter the physicochemical properties of calcium phosphate, but also have their own significances in the biological response. For instance, strontium as one of bone-seeking elements is distributed in bone, and plays an important biological role in
M. Huang et al. / Materials Chemistry and Physics 147 (2014) 540e544
human bone metabolic turnover [18,19]. It is generally accepted that Sr2þ can replace Ca2þ in the crystal lattice within a large concentration range owing to the chemical similarity. Its substitution results in the crystal destabilization and then the increasing solubility [20e22]. Furthermore, the low dose of Sr substitution has the beneficial influence on the decrease of bone resorption and the enhancement of bone formation by inhibiting osteoclast-resorbing function and stimulating osteoblast activity and differentiation [11,23,24]. Therefore, this work is focused on the doping of Sr in TCP to form Sr-TCP using a new biotemplating approach. Various synthetic methods for Sr-TCP powders have been intensively studied, such as the solid state calcination, coprecipitation, solegel process, and hydrothermal method [20,25e27]. There are commonly two ways, the solid-phase and liquid phase method. In the former method, the solid-phase Sr salts incorporate with calcium and phosphate salts to form Sr-TCP at a high sintering temperature [25]. However, it is quite difficult to completely mix the raw materials, and a subsequent milling process is necessary. Thus the product morphology and the Sr distribution have a poor controllability. By a liquid phase method, Sr2þ is incorporated into the amorphous calcium phosphate structure during the precipitation process, and then doped into the crystal lattice after calcinations, which is confirmed as a more effective way to obtain Sr-TCP particles. However, most traditional liquid phase method can only prepare the sheet/rod-like powders without any additives. The effective control over the composition, structure and morphology is still a challenge. In terms of the increasing scientific interest towards the biomimetic synthesis and the recognition for microspheres [28e32], we, along with others, have developed a biotemplate method which proves to be feasible in the fabrication of functional biomaterials [33e35]. Moreover our previous works have successfully prepared a calcium phosphate microcapsule via a biomimetic mineralization process using yeast cells as a template core [34]. In this work, we aim at developing an effective way to synthesize porous Sr-TCP microspheres via the yeast-based biotemplate method. The Sr dose in the microspheres can be effectively controlled. Moreover, the biocompatibility of Sr-TCP was evaluated. The final product was proposed to have potential applications in bone medical engineering.
2. Materials and methods 2.1. Synthesis Yeast cells (Saccharomyces cerevisiae) were isolated and purified from the ANGEL's instant dry yeast by the spread plate method and the streak plate method. They were cultured in YPD medium (1% yeast extract, 2% peptone, and 2% glucose), incubated at 28 C with shaking at 180 rpm. The synthesis of Sr-TCP microspheres was described in the reference [34]. Yeast cells were alternately immersed into Poly(diallyldimethylammonium chloride) (PDADMAC) and Poly(acrylic acid) (PAA) solution via the layer-by-layer treatment (LBL) and formed yeast/PDADMAC/PAA composite biotemplates. The biotemplates were mixed into calcium chloride (CaCl2) and strontium chloride (SrCl2) solution with different Sr/ (Sr þ Ca) molar ratios, stirring for 12 h. The biotemplates absorbed Sr2þ and Ca2þ were added to the diammonium phosphate ((NH4)2HPO4) solution, stirring for 12 h under ambient temperature. The pH value was regulated about 10. After centrifugation and washed, the product was dried and then calcined at 800 C. The corresponding products with different Sr/(Sr þ Ca) molar ratios (0, 1, 5, 10 and 15%) were denoted as Sr0, Sr1, Sr5, Sr10 and Sr15, respectively.
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2.2. Cell cytotoxic experiment The initial cell of the 3rd generation of human marrow stromal cells (hMSCs) was donated by the First Affiliated Hospital of Sun Yat-sen University in Guangzhou as described in the reference [36]. Cells were exposed to Sr-TCP powders via the following way [37]. Sr-TCP powders were autoclaved and stored in a sealed tube. The sample was re-suspended with the low sugar complete medium using ultrasound to ensure a predetermined final concentration (200 mg ml1). The cell suspension with the 7th generation of hMSCs was added into the suspension of Sr-TCP powder, using a cell counter counts to reach a specified concentration range (4 103 cells/hole). The mixture was feed in 96-well plates and placed in incubator (37 C, 5% CO2). The Cell Counting Kit-8 (CCK-8) was used to test the cell proliferation/toxicity experiment. Tetrazolium salt WST®-8 under the effect of 1-Methoxy PMS electron carrier is reduced to a watersoluble yellow formazan dye. The toxicity was estimated with measuring the absorbance of the solution. The absorbance value at 450 nm represents the number of living cells. The samples without powders and the one with a 0.65% phenol solution were regarded as the negative control and positive control, respectively. All samples were assayed at the time points (4 h, 1 day, 3 day and 7 day). 2.3. Characterization X-ray diffraction (XRD) patterns of samples were obtained on a Panalytical X'Pert PRO (PANalytical, Netherlands) using Cu Ka (l ¼ 0.15418 nm) radiation. The morphologies of the products were observed by the scanning electron microscopy (SEM, Hitachi H-800). The elements qualitative and semi-quantitative analysis of the sample was tested using the electron spectrometer (EDS, EPMA-1600, Shimadzu Corporation). The CCK-8 assay was detected using the multifunctional microplate reader (Thermo3001, U.S. Thermo Electro Corporation). 3. Result and discussion Fig. 1 shows morphologies of yeast cells before and after mineralization, as well as porous Sr-TCP microspheres calcined at 800 C. The yeast-based biotemplates method was proved to be an effective way to prepared calcium phosphate microspheres. Yeast cells were shrivelled after the freeze dry treatment with a soft surface (Fig. 1A1eA3). After the mineralization, particles appeared to be plump and presented a complete spherical structure with a diameter about 5 mm (Fig. 1B1eB2). Numbers of cluster-like protuberances appeared on the rough surface (Fig. 1B3). The microsphere structure remained after calcination at 800 C (Fig. 1C1eC3). Crystal grains were neck-like connected and made up of the porous shell. The fine nano-scale pores on the surface of the microsphere have an average diameter of 120 ± 14 nm based on the measurement from SEM photos. In addition, the microsphere had a hollow structure resulting from the biotemplate removing under heat treatment. From some broken microspheres, the porosity was estimated with a simple spherical model. The diameter of the microsphere and the thickness of shell were measured on SEM photos. The average porosity of microsphere was about 70% ± 4% (v/v) (n ¼ 10), which was calculated by using the equation
P¼
10 1 X Di 2di 3 100% 10 i¼1 Di
(1)
where Di is the spherical diameter of the ith microsphere, di is the thickness of the ith shell, and P is the average porosity of
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Fig. 1. Morphologies of yeast cells before and after mineralization as well as Sr-TCP microspheres calcined at 800 C. A1-A3 represents the different magnifications of dry yeast cells via the freeze drying treatment. B1eB3 represents the different magnifications of Sr-TCP microspheres prepared by using the yeast-based biotemplates method before calcined. C1eC3 represents the different magnifications of Sr-TCP particles calcined at 800 C.
microsphere. Compared to the TCP microcapsule without Sr2þ addition (Seen in Support Information), the protuberances before calcination and smaller nanocrystals after calcination on the microsphere surface may result from the Sr2þ incorporation in TCP. The Sr2þ substitution can affect the TCP crystal lattice and lead to the morphological change [21]. Fig. 2 shows the XRD pattern of the products obtained with different original Sr/(Sr þ Ca) ratios. According to the JCPDS database 00-009-0169, the b-TCP phase (Ca3(PO4)2, whitlockite) was present in all samples (Fig. 2B). However, with the Sr/(Sr þ Ca) ratios increasing from 0 to 15 at%, XRD patterns gradually shifted to the lower angle, and the diffraction intensity of main characteristic peaks at 31.0 , 27.8 and 34.4 weakened (Fig. 2A). These results suggested that Sr2þ partly replaced Ca2þ in TCP crystal lattice
[38,39]. Since the Sr2þ radius (1.13 Å) is larger than Ca2þ (0.96 Å), the difference of ionic radius caused part of the lattice distortion. Lattice parameters (a and c-axis) as well as the unit cell volume linearly increase with Sr-addition [39]. The diffraction angle and the characteristic peak intensity were thus slightly altered. The Sr substitution can hinder the normal grain growth, which further changed the surface morphology of TCP microspheres (shown in Fig. 1). Besides, the shift degree directly related to the Sr content in particles which was tested in Fig. 3. Fig. 3 shows the EDS analysis of Sr-TCP powders with different original Sr/(Sr þ Ca) ratios. Elements of Ca, P, O and C were clearly shown (Fig. 3B). Ca, P and O elements belonged to TCP, while C elements may be derived from the CO2 in surrounding environment or a small amount of CO2 3 substitution during calcinations [34].
Fig. 2. XRD patterns of Sr-TCP powders calcined at 800 C. Samples denoted as Sr0, Sr1, Sr5, Sr10 and Sr15 represents the product derived from different original ratios of Sr/ (Sr þ Ca) at 0, 1, 5, 10 and 15 at%, respectively. A was the magnified version of the shadowed area in B.
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Fig. 3. EDS analysis of Sr-TCP products calcined at 800 C with different original Sr doses represented by the original ratios of Sr/(Sr þ Ca). A represents the atomic percentage of Sr to Sr þ Ca in Sr-TCP calculated from EDS spectra shown in B. B represents the EDS analysis for all elements of Sr-TCP.
Particularly, the Sr characteristic peak emerged with Sr-addition. Its intensity increased linearly with the original Sr/(Sr þ Ca) ratios gradually increasing from 0 to 15 at% (Fig. 3A). The atomic percentage of Sr to Sr þ Ca in Sr-TCP showed a linear incorporation of Sr through the substitution of Ca in TCP lattice. The increasing Sr content may lead to the shift of diffraction peaks in XRD patterns. The result showed that the Sr content in TCP microspheres can be effectively controlled via the regulation of original Sr/(Sr þ Ca) ratios. Moreover, Sr2þ released slowly in a long time when Sr-TCP powders immerged in the phosphate buffered saline (seen in Support information). Fig. 4 shows morphologies of hMSCs cultured with Sr-TCP powders at 7 day. The fusiform living cells evenly overspread on the plate bottom without adding powders (Fig. 4A). However, the
cells' distribution was disturbed after adding Sr-TCP powders (Fig. 4B). Cells were inclined to be an aggregation. From light field optical images, particles were observed to be a banding-like distribution (Fig. 4C), which may result from the particle adhesion onto the cell surface. Block particle aggregations were present (Fig. 4D). We believe that the block can provide the better field for cell adhesion and conduct the cell growth to the three-dimensional space. Thus cells densely adhered and climbed around block aggregations, which may result in the appearance of cells' aggregations in Fig. 4B. The result indicates that the Sr-TCP powders have an excellent biocompatibility with hMSCs. Further demonstration about Sr-TCP biocompatibility was shown in Fig. 5. The CCK-8 assay result indicated that OD450nm values of all experimental samples significantly increased with the
Fig. 4. Morphologies of hMSCs cultured with Sr-TCP powders at day 7. A represents the fluorescent image from the hMSCs sample without adding Sr-TCP powders. B represents the fluorescent image from the hMSCs sample after interaction with Sr-TCP powders. Living cells show green emission. C and D represent the different areas of the bright field optical image taken from the hMSCs sample after interaction with Sr-TCP powders. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.matchemphys.2014.05.028.
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
[10] Fig. 5. The CCK-8 analysis from controls and experimental hMSCs samples after cocultured with Sr-TCP powders for 4 h, 1 d, 3 d and 7 d. Positive and negative control were obtained from the sample with 0.65wt% phenol solution and the blank plate. (&, # and Ф indicated statistical significance when the experiment group was compared with the positive control, negative control, and the sample Sr0 without adding Sr2þ, respectively, p < 0.05).
prolonged incubation time. Compared to the positive control, all SrTCP powders showed statistically significant (p < 0.05) promotion on the hMSCs proliferation. Except the sample Sr1, OD450nm values of the rest experimental samples indicated statistical significance compared with the negative control and Sr0 (p < 0.05) after 7 days' culture. The result indicated the proliferation rate of hMSCs cocultured with Sr-TCP powders is always higher than that with pure TCP, even higher than the negative control in blank plate. Therefore, Sr-TCP powders prepared via yeast-based biotemplate method were confirmed to be beneficial for the hMSCs proliferation. 4. Conclusions
[11] [12]
[13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
A yeast-based biotemplating method was introduced for the effective synthesis of Sr-TCP microspheres. The porous Sr-TCP microsphere with a diameter of approximately 5 mm was obtained by allowing the biotemplate to pre-adsorb a mixture solution of Sr2þ and Ca2þ with different original Sr/(Sr þ Ca) molar ratios. The pre-adsorption enables the doping of Sr2þ in TCP crystal lattice by partially substituting Ca2þ. The amount of Sr doped in TCP can be effectively controlled. The Sr-TCP powder exhibited an excellent biocompatibility with hMSCs. We propose the final product can be used as a drug carrier and applied in the medical field for the bone regeneration. Acknowledgements This work was supported by National Basic Research Program of China (2012CB619100), National Nature Science Foundation of China (50732003, U0834003, 51072056), and Program for Changjiang Scholars and Innovative Research Team in University (IRT 0919).
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