Osteoprecursor cell response to strontium-containing hydroxyapatite ceramics Weichang Xue,1 Jessica L. Moore,3 Howard L. Hosick,1,3 Susmita Bose,1,2 Amit Bandyopadhyay,1,2 W.W. Lu,4 Kenneth M.C. Cheung,4 Keith D.K. Luk4 1 Bioengineering Research Center, Washington State University, Pullman, Washington 99164 2 School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164 3 School of Biological Sciences, Washington State University, Pullman, Washington 99164 4 Department of Orthopaedic and Traumatology, The University of Hong Kong, Hong Kong, China Received 30 December 2005; revised 21 February 2006; accepted 28 February 2006 Published online 2 August 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30815 Abstract: The objective of this study was to investigate the in vitro bioactivity of strontium-containing hydroxyapatite (Sr-HA), and its effect on cellular attachment, proliferation, and differentiation. The effect of Sr-HA has been compared with that of hydroxyapatite (HA), which is widely used in orthopedics and dentistry. Sr-HA ceramic containing 10 mol % was prepared. The bioactivity of SrHA was evaluated in vitro by immersion in simulated body fluid (SBF). After immersion in SBF, Sr-HA exhibited greater ability to induce apatite precipitation on its surface than did HA. The possible effects on cell behavior of SrHA were examined by culturing osteoprecursor cells (OPC1) on materials surfaces. Cell shape and cell-material interactions were analyzed by scanning electron micro-
scope (SEM) and the MTT assay was used to determine cell proliferation on samples. When compared with HA, Sr-HA promoted better OPC1 cell attachment and proliferation, and showed no deleterious effects on extracellular matrix formation and mineralization. Confocal scanning microscopy was used to assess the expression of specific osteoblast proteins: alkaline phosphatase (ALP) and osteopontin (OPN). The results obtained indicate that the presence of Sr stimulates OPC1 cell differentiation, and enhances ALP and OPN expression. Ó 2006 Wiley Periodicals, Inc. J Biomed Mater Res 79A: 804–814, 2006
INTRODUCTION
humans, numerous clinical studies have shown that strontium appeared to be a useful therapeutic agent for the treatment of postmenopausal osteoporosis.6–8 In these studies, strontium administration to postmenopausal osteoporotic women resulted in a significant increase in bone mass and bone strength by a dual mechanism of action: inhibition of bone resorption and augmentation of bone formation. Although information regarding the effects of strontium on bone cells in vitro is scant, some studies have shown that strontium enhances the replication of preosteoblastic cells, and stimulates bone formation in cell and calvarial cultures.9 It also has been demonstrated that strontium ranelate decreases bone resorption in vitro by inhibiting osteoclast activity.10 However, the cellular and molecular mechanisms of action of strontium on bone cells are yet to be understood. The current studies on the biological role of strontium mainly focus on strontium ranelate. Recently, strontium-substituted calcium phosphates have also been developed.11–18 Calcium phosphate ceramics, particularly hydroxyapatite (HA) and tricalcium
The role of strontium (Sr) in human pathology has attracted less attention than the other two important divalent metals, calcium and magnesium. However, there has been an increasing awareness of the biological role of strontium since the development of the drug strontium ranelate, which has recently been shown to reduce the incidence of fractures in osteoporotic patients.1,2 There is growing evidence that strontium has a beneficial effect on bone.3–5 The currently available data indicate that strontium administration at low dose reduces bone resorption and increases bone formation, resulting in increased bone mass in normal or ovariectomized animals.3–5 In
Correspondence to: S. Bose; e-mail:
[email protected] Contract grant sponsor: Office of Naval Research; contract grant numbers: N00014-1-04-0644 and N00014-1-050583 ' 2006 Wiley Periodicals, Inc.
Key words: strontium; hydroxyapatite; bioactivity; osteoblast
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Figure 1. SEM micrographs of (a) HA and (b) Sr-HA samples after sintering. Note the similar grain size and microstructure of two samples.
phosphate (TCP), have been widely used in orthopedics and dentistry because of their excellent biocompatibility with human hard tissues.19,20 On the basis of their chemical resemblances, partial Ca2+ in HA can be replaced by Sr2+. Several methods have been introduced to fabricate strontium-substituted calcium phosphate ceramics and bone cements.11,12 The structural changes, growth and dissolution behavior, and mechanical properties of these materials were also studied. It was reported that the replacement of partial Ca2+ in HA by Sr2+ can change material dissolution behavior and growth kinetics.13 The mechanical properties of HA were reportedly improved by incorporating 5 mol % Sr instead of equivalent Ca.14 Strontium-containing HA has also been developed as a bioactive bone cement.15–18 However, most of these studies are restricted to the fabrication and structural investigation of the materials without a detailed evaluation of biological performance. The objective of this study is to investigate the bioactivity of strontium-containing hydroxyapatite (SrHA) in vitro, and its effect on cellular attachment, proliferation, and differentiation. The effect of Sr-HA is compared with that of HA, which is already being widely used in orthopedics and dentistry.
and Sr-HA powder was precipitated by the process as follows:
10 CaðOHÞ2 þ 6 H3 PO4 ! Ca10 ðPO4 Þ6 ðOHÞ2 þ 18H2 O 10 SrðOHÞ2 þ 6 H3 PO4 ! Sr10 ðPO4 Þ6 ðOHÞ2 þ 18H2 O The powder obtained was pressed into disc form (Ø10 3 mm2) by uniaxial pressing with a pressure of 10 MPa. Sr-HA samples were sintered at 12508C for 3 h. As a control material, HA (Barkley Advanced Materials, CA) samples were also prepared by the same procedure. After gold coating, the samples surfaces were observed under a scanning electron microscope (SEM, Hitachi s-570, Japan).
Mineralization in simulated body fluid The bioactivity of Sr-HA was evaluated in vitro by immersion in simulated body fluid (SBF) (2.5 mM of Ca2+, 1.5 mM of Mg2+, 142.0 mM of Na+, 5.0 mM of K+, 148.5 mM of Cl, 4.2 mM of HCO3, 1.0 mM of HPO42, 0.5 mM of SO42).20 The SBF solution was buffered at pH 7.40 with 50 mM tri (hydroxymethyl) aminomethane ((CH2OH)3CNH2) and approximately 45 mM hydrochloric acid (HCl) at 378C. After immersion for 3 and 14 days, the surface microstructures of the samples were observed with SEM.
Cell culture MATERIALS AND METHODS Sample preparation and characterization Sr-HA powder containing 10 mol % Ca2+ replaced by Sr2+ was synthesized as stated in a previous work.17 Ca(OH)2, Sr(OH)2, and H3PO4 were used as the precursors
All samples were sterilized by autoclaving at 1218C for 20 min. In this study the cells used were an immortalized, cloned osteoblastic precursor cell line 1 (OPC1), which was derived from human fetal bone tissue.21 OPC1 cells were seeded onto the samples and then placed in 24-well plates. Cell density was 2.0 104 cells/well. 1 mL of McCoy’s 5A medium (enriched with 5% fetal bovine serum, 5% bovine calf serum and supplemented with 4 mg/mL of fungizone) Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
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Figure 2. Surface micrographs of the samples after immersion in SBF: (a) HA after 3 days, some apatite granules precipitated randomly on the surface. (b) HA after 14 days, (c) Sr-HA after 3days, and (d) Sr-HA after 14 days of immersion. Note a new-formed apatite layer completely covered sample surfaces. was added to each well. Cultures were maintained at 378C under an atmosphere of 5% CO2. Medium was changed every 2–3 days for the duration of the experiment. Samples for testing were removed from culture at 3, 7, and 14 days of incubation.
MTT assay The MTT assay (Sigma, St. Louis, MO) was performed to assess cell proliferation. The MTT solution of 5 mg/mL was prepared by dissolving MTT in PBS, and filter sterilized. The MTT was diluted (50 mL into 450 mL) in serum free, phenol red-free Dulbeco’s Minimum Essential medium (DME). 500 mL diluted MTT solution was then added to each sample in 24-well plates. After 2 h incubation, Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
500 ml of solubilization solution made up of 10% Triton X100, 0.1N HCl and isopropanol were added to dissolve the formazan crystals. 100 mL of the resulting supernatant was transferred into a 96-well plate, and read by a plate reader at 570 nm. Data are presented as mean 6 standard deviation. Statistical analysis was performed using Student’s ttest, and p < 0.05 was considered significant.
Morphology of OPC1 cells on samples All samples for SEM observation were fixed with 2% paraformaldehyde/2% glutaraldehyde in 0.1M cacodylate buffer overnight at 48C. Post-fixation was performed with 2% osmium tetroxide (OsO4) for 2 h at room temperature. The fixed samples were then dehydrated in an ethanol se-
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Figure 3. SEM micrographs of OPC1 cells after 3 days of culture on HA (a,c), exhibited an elongated, flattened morphology with fewer filopodia extensions. On Sr-HA (b,d), more cells grew on the surface, with many lamellipodia and filopodia extensions. ries (30, 50, 70, 95, and 100% three times), followed by a hexamethyldisilane (HMDS) drying procedure. After gold coating, the samples were observed by SEM.
Immunocytochemistry and confocal microscopy Samples bearing cells were fixed in 4% paraformaldehyde in 0.1M phosphate buffer. Those samples were stored at 48C, for future use. After rinsing in Triton for 10 min, samples were blocked with TBST/BSA (tris-buffered saline with 1% bovine serum albumin, 250 mM NaCl, pH 8.3) for 1 h. Primary antibody against alkaline phosphate (ALP) (Sigma, St. Louis, MO) or osteopontin (OPN) (Chemicon International, Temecula, CA) was added at a 1:100 dilution and incubated at room temperature for 2 h. The secondary
antibody, goat anti-mouse (GAM) Oregon green (Molecular Probes, Eugene, OR), was added at a 1:100 dilution and incubated for 1 h. Samples were then mounted on coverslips with Vectashield Mounting Medium (Vector Labs, Burlingame, CA) with propidium iodide (PI) and observed using a confocal scanning laser microscopy (BioRad 1024 RMC).
RESULTS Sample microstructure Typical microstructures of Sr-HA and HA sintered at 12508C are shown in Figure 1. Two samples are almost fully dense, and display similar grain size Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
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Figure 4. SEM micrographs of OPC1 cells on (a) HA and (b,c) Sr-HA after 7 days of culture. Note extracellular matrix (ECM) on the surface of Sr-HA.
and microstructure. The average grain size of HA and Sr-HA was 8.3 mm and 9.1 mm, respectively. There is no evidence that the incorporation of Sr has significant effect on HA microstructure. Mineralization behavior in SBF Mineralization behavior of Sr-HA was investigated in vitro by immersion in SBF solution. Figure 2 shows the surface micrographs of the samples after immersion for 3 and 14 days, respectively. After 3 days of immersion, Sr-HA surface appeared to be covered by a newly formed layer, which was chemically similar to apatite based on EDS analysis. Some apatite granules also were found on the surface of Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
Sr-HA [Fig. 2(c)]. In comparison, apatite granules also precipitated randomly on the HA surface, but they did not cover all of the surface of HA [Fig. 2(a)]. The results at day 3 indicate that Sr-HA has greater efficiency to induce apatite formation early on its surface than does HA. After 14 days of immersion, both surfaces were completely covered with a newly formed apatite layer [Fig. 2(b,d)] and no significant differences could be observed between the two samples. Morphology of OPC1 cells on samples OPC1 cells on sample surfaces were analyzed for cell shape and cell-material interactions using SEM.
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Figure 5. SEM micrographs of OPC1 cells on (a,c) HA and (b,d) Sr-HA after 14 days of culture. The samples surfaces were completely covered by a dense and confluent cellular multilayer that deposited an abundant amount of ECM, forming a three-dimensional fibril network.
Figure 3 shows SEM morphologies of OPC1 cells on HA and Sr-HA after 3 days of culture. OPC1 cells attached well on both Sr-HA and HA. However, when compared with Figures 3a and 3b, more cells were observed on Sr-HA. OPC1 cells cultured on SrHA exhibited a typical osteoblast phenotype, which appears cuboidal and three-dimensional, with many lamellipodia and filopodia extensions [Fig. (3d)]. Filopodia are noted contacting cells with substrate in addition to neighboring cells. Cells on HA exhibited an elongated, flattened morphology, as shown in Figure 3c. Fewer filopodia between cells and HA were evident when compared with that between cells and Sr-HA.
After 7 days of culture, cells had undergone a significant spreading on the surface of the two scaffold types [Figs. 4(a,b)]. More cells could be observed on scaffolds than at day 3. Cells on Sr-HA started to grow to confluence. In general, fewer cells grew on the surface of HA when compared to that of Sr-HA. Early stage extracellular matrix (ECM) formation was found on the surface of cells or between the neighboring cells grown on Sr-HA.17 The ECM is full of fibers that appear to be collagen [Fig. 4(c)]. SEM images of OPC1 cells cultured for 14 days are presented in Figure 5. The surface of Sr-HA was completely covered by a dense and confluent cellular multilayer that deposited an abundant amount of Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
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Figure 6. MTT assay of cells on HA and Sr-HA. The number of cells increased with the increase of culture time. The number of cells on Sr-HA was more than those on HA. (*p < 0.05, **p < 0.01) [Color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.]
ECM, forming a three-dimensional fibril network [Fig. 5(d)]. Some mineralized ECM was also observed on Sr-HA. The number of cells grown on HA appears to be less than that on Sr-HA, but the presence of ECM could also be detected on the surface of the cells [Fig. 5(c)]. MTT assay The MTT assay was used to determine OPC1 cell proliferation on HA and Sr-HA. Fig. 6 shows a comparison of cell densities on two scaffold types over the course of the experiment. Cell proliferation was evident over the duration of the experiment, both on HA and on Sr-HA. After 14 days of culture, the number of cells increased approximately 15 times for Sr-HA and 9 times for HA, in relation to day 3. Data from the MTT assay also show the difference between the two scaffolds. After 3 days of culture, Sr-HA had higher cell density, which was approximately 3 times that on HA. The difference between the two scaffolds became smaller with the increase of culture time. However, the number of cells on SrHA was always more than those on HA. Immunocytochemistry and confocal microscopy Immunocytochemistry of OPC1 cells was used to determine whether the cells express an osteoblastic phenotype on samples. Because a major characteristic of osteoblasts is the expression of ALP, immunostaining viewed on the confocal microscope identiJournal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
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fied the presence of ALP is expected. Figure 7 shows the confocal micrographs of ALP expression in OPC1 cells cultured on HA and Sr-HA. ALP within the cells is identified by the expression of green fluorescence and nuclei counterstained with PI in the mounting medium expressed red fluorescence. At all time points, OPC1 cells cultured on both HA and SrHA displayed positive immunostaining for ALP, but with different patterns and levels. After 3 days of culture, cells on Sr-HA showed obvious ALP production [Fig. 7(d)], whereas only small signal could be detected on HA [Fig. 7(a)]. With the increase of culture time, ALP activity increased significantly. The results at day 7 and day 14 also exhibit the difference between the two scaffolds. ALP expression on HA was always lower than on Sr-HA. Confocal micrographs of OPN expression in OPC1 cells cultured on HA and Sr-HA are shown in Fig. 8. OPN within the cells is identified by the expression of green fluorescence and nuclei counterstained with PI expressed red fluorescence. OPN expression also exhibits different patterns and levels on two different scaffolds. At day 3, cells on Sr-HA show obvious OPN expression [Fig. 8(d)], but a negligible signal can be detected on HA [Fig. 8(a)]. At day 7, OPN activity exhibited a significant increase on both scaffolds. After 14 days of culture, the OPN expression appeared to slightly decrease when compared with day 7. The difference between the two scaffolds decreased with increase in time. DISCUSSION This study was focused on understanding the bioactivity of Sr-HA in SBF, and its effect on proliferation, cellular morphology, and ALP and OPN activity of OPC1 cells in vitro. Also, performance of SrHA was compared with that of HA, which is widely used in orthopedics and dentistry. Studies of the bone-biomaterials interface reveal that a common characteristic of bioactive materials is the consistent presence of an interfacial apatite layer.22 This can be reproduced in vitro by immersion experiments using a simulated physiological solution that mimics the typical ion concentration in the body. In this study, Sr-HA exhibited the ability to induce apatite formation on its surface only after 3 days in SBF. The fact of apatite formation on SrHA indicates that this ceramic has good bioactivity. Sr-HA exhibits greater ability to induce apatite formation on its surface than does HA, after 3 days of immersion. Our results suggest that this is the direct result of a higher dissolution rate of Sr-HA. It has been demonstrated that the replacement of partial Ca2+ in HA by Sr2+ can change dissolution behavior and growth kinetics. Christoffersen and his co-work-
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Figure 7. Confocal micrographs of ALP expression in OPC1 cells cultured on (a) HA at day 3, (b) HA at day 7, (c) HA at day 14, (d) Sr-HA at day 3, (e) Sr-HA at day 7, and (f) Sr-HA at day 14. Green fluorescence indicating antibody bound to ALP, red fluorescence indicating antibody bound to DNA (nucleus). Bar ¼ 100 mm. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]
ers13 investigated the dissolution behavior of HA containing 1–10 mol % Sr2+ and found an increase of the solubility of these apatites with increase in Sr2+ content. Chen et al.23 proposed that the incorporation of low-dose strontium introduces more lattice
distortions into the structure of hydroxyapatite and thus increases its solubility. The dissolution of SrHA results in the release Ca2+, which increases the ionic activity product of the apatite in surrounding fluid, and thus readily induces apatite precipitation. Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
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Figure 8. Confocal micrographs of OPN expression in OPC1 cells cultured on (a) HA at day 3, (b) HA at day 7, (c) HA at day 14, (d) Sr-HA at day 3, (e) Sr-HA at day 7, and (f) Sr-HA at day 14. Green fluorescence indicating antibody bound to OPN, red fluorescence indicating antibody bound to DNA (nucleus). Bar ¼ 100 mm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
The development of OPC1 cell on the material is characterized by a sequence of events, involving cell adhesion, proliferation, ECM biosynthesis, ECM development and maturation, and ECM mineralization.24 Cell adhesion and spreading belong to the Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
first phase of cell–material interactions, and are also probably the most critical stage. The quality of this first phase will influence the cell’s capacity to proliferate and differentiate.25 The MTT result showed that, after 3 days of culture, the number of cells on
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Sr-HA was approximately 3 times more than those on HA. This result suggests that the presence of Sr enhances cellular attachment. In support of this claim, it was observed that filopodia extensions from the cells to the substrate are more abundant on SrHA than HA. In addition to communication between cells, external physical features such as filopodia and microextensions are also used in attachment and migration.25 The cell–material interaction depends on the surface aspects of materials, which include topography, chemistry, and surface energy.25 Because of the similar surface topography between Sr-HA and HA, the improvement of cellular attachment on Sr-HA can be ascribed to the presence of Sr. In addition, as previously described, Sr-HA has a higher dissolution rate than HA. The subsequently roughened surface microtopography of Sr-HA due to rapid dissolution may also promote cellular attachment and spreading.26 After attachment phase, cells grow continually to reach confluence. The MTT assay and SEM observation showed that Sr-HA is not cytotoxic, and thus does not inhibit cell proliferation. In fact, more cells were observed on Sr-HA than on HA during the experiment. The cells on Sr-HA grow to confluence only after 7 days of culture, which occurs later on HA. These findings imply that Sr has a beneficial effect on cell proliferation. During the proliferation period, these cells also synthesize ECM, which is mainly composed of collagen. The presence of collagenous ECM indicates that OPC1 cells on Sr-HA are capable of forming a matrix suitable for mineralization, and suggest initiation along a biomineralization pathway. SEM observation showed that ECM forms relatively early on Sr-HA, suggesting that Sr-HA accelerates cell differentiation at an early stage and promotes ECM formation. This result is consistent with previous study that Sr stimulates collagen synthesis.27 Literature also indicate that a high intake of strontium may disturb bone mineralization.28 Strontium may replace calcium in bone, causing distortion of the crystal lattice, impairing crystal growth, and increasing dissolution of mineralized bone. In this study, mineralized ECM appeared on Sr-HA, indicating that the presence of Sr in HA did not induce deleterious effects on matrix mineralization. This may be due to the fact that Sr dissolved from Sr-HA at a low level, which may not diminish mineralization.29 At a low level, long-term treatment with Sr increased collagenous matrix formation without inducing deleterious effects on matrix mineralization.27 Associated with proliferation and differentiation, OPC1 cells express a number of osteoblastic phenotypic markers. ALP is a major characteristic marker of osteoblasts. ALP is regarded as an early marker for osteoblast differentiation, and it is generally
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accepted that as the specific activity of ALP in a population of bone cells increases there is a corresponding shift to a more differentiated state.30 ALP expression appears strongly positive in differentiating osteoblastic cells. High levels of ALP expression on Sr-HA, especially at early time points, could be related to faster differentiation of OPC1 cells. In addition, ALP has also been proposed to play a role in the initiation of matrix mineralization.31 Enhancement of ALP expression on Sr-HA at day 14 indicates that Sr-HA has a beneficial effect on ECM mineralization. Of the non-collagenous proteins, OPN is a secreted phosphoglycoprotein isolated originally from the extracellular matrix of bone, and it is synthesized by osteoblastic cells. OPN is likely to play a role in attachment and migration of osteoblasts during the early formative stages of osteogenesis.31 Therefore, higher levels of OPN expression on Sr-HA at early time points suggest that Sr-HA may promote cell attachment and migration, which is consistent with SEM observations. OPN is expressed during the period of active proliferation, decreases post-proliferation, and then exhibits induction at the onset of mineralization, achieving peak levels of expression paralleling the accumulation of mineral.24 OPN expression slightly decreased at day 14 when compared with that at day 7. Our data suggest that at day 14, OPC1 cells are at a transitional stage between the end of proliferation and the beginning of mineralization. Enhancement of cellular function on Sr-HA can be attributed to the high solubility of Sr-HA and the beneficial stimulation of Sr on cells. The addition of Sr increases the solubility HA and also readily induces the precipitation of apatite. Bone-like apatite formation on surfaces is helpful to cell attachment and proliferation. In addition, promoting calcium release from Sr-HA is also suggested to activate calcium channels and thus stimulate cell response.32 In addition to the change of solubility, the presence of Sr is another important reason for enhancement of the cellular function of Sr-HA. Although the detailed cellular and molecular mechanisms of strontium effects on bone cells have still to be determined, numerous studies1–10 in vivo and in vitro have demonstrated that Sr promotes osteoblast function and subsequent bone formation. Therefore, it is reasonable to believe that Sr, either in Sr-HA ceramics or dissolved into surrounding media, has a beneficial effect on OPC1 cells.
CONCLUSIONS We have studied the bioactivity of Sr-HA ceramics in comparison with phase pure HA. Sr-HA ceramic Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
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exhibited high bioactivity in SBF, which was clear from fast apatite formation on its surface. Cell culture test indicated that Sr-HA has good biocompatibility with human osteoblast. Compared with HA, Sr-HA promoted OPC1 cell attachment and proliferation, and exhibited no deleterious effects on ECM formation and mineralization. It also has been demonstrated that the presence of Sr stimulates OPC1 cell differentiation, and enhances ALP and OPN expression. It is clear from our study that Sr promotes osteoblast function and subsequent bone formation. Further research is needed to understand detailed cellular and molecular mechanisms of strontium effects on bone cells. The authors gratefully acknowledge experimental help from Drs. Chris Davitt and Valerie Lynch-Holm in association with the Electron Microscopy Center at Washington State University.
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