A novel strontium(II)-modified calcium phosphate bone cement stimulates human-bone-marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation in vitro

Acta Biomaterialia 9 (2013) 9547–9557

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A novel strontium(II)-modified calcium phosphate bone cement stimulates human-bone-marrow-derived mesenchymal stem cell proliferation and osteogenic differentiation in vitro M. Schumacher a, A. Lode a,⇑, A. Helth b, M. Gelinsky a a b

Centre for Translational Bone, Joint and Soft Tissue Research, Medical Faculty and University Hospital, Technische Universität Dresden, Dresden, Germany Leibniz-Institute for Solid State and Materials Research IFW Dresden, Dresden, Germany

a r t i c l e

i n f o

Article history: Received 15 March 2013 Received in revised form 22 July 2013 Accepted 23 July 2013 Available online 31 July 2013 Keywords: Calcium phosphate bone cement Strontium Cell biological evaluation Human mesenchymal stem cells Osteoblasts

a b s t r a c t In the present study, the in vitro effects of novel strontium-modified calcium phosphate bone cements (SrCPCs), prepared using two different approaches on human-bone-marrow-derived mesenchymal stem cells (hMSCs), were evaluated. Strontium ions, known to stimulate bone formation and therefore already used in systemic osteoporosis therapy, were incorporated into a hydroxyapatite-forming calcium phosphate bone cement via two simple approaches: incorporation of strontium carbonate crystals and substitution of Ca2+ by Sr2+ ions during cement setting. All modified cements released 0.03–0.07 mM Sr2+ under in vitro conditions, concentrations that were shown not to impair the proliferation or osteogenic differentiation of hMSCs. Furthermore, strontium modification led to a reduced medium acidification and Ca2+ depletion in comparison to the standard calcium phosphate cement. In indirect and direct cell culture experiments with the novel SrCPCs significantly enhanced cell proliferation and differentiation were observed. In conclusion, the SrCPCs described here could be beneficial for the local treatment of defects, especially in the osteoporotic bone. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Osteoporosis is a systemic disease ‘‘characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk’’ [1], which results from an impaired balance of bone resorption and formation by osteoclasts and osteoblasts, respectively. For the year 2000, the number of primary osteoporosis-related fractures was estimated to be 9 million worldwide, with 61% of fractures occurring in women [2]. Considering the worldwide demographic development, the burden of osteoporosis will further increase in the near future. Various therapies have been developed for the clinical treatment of osteoporosis based on either the inhibition of bone resorption (e.g. by bisphosphonates, strontium ranelate SrR, RANKL antibody denosumab) or the anabolic stimulation of bone formation (e.g. by parathyroid hormone peptides, SrR) [3,4]. The impact of strontium(II), acting both as an inhibitor of resorption as well as a stimulus of bone formation, has already been demonstrated in vitro and in vivo [4–6]. Strontium was shown to affect cellular processes via the membrane-bound calcium sensing receptor (CaSR), not only in osteoblasts but also in cells of the osteoclasts lineage, where CaSR interaction with ⇑ Corresponding author. E-mail address: [email protected] (A. Lode).

strontium can inhibit pre-osteoclast maturation and induce apoptosis in mature osteoclasts [7,9,10]. Mesenchymal stem cell as well as pre-osteoblast proliferation and differentiation into bone-forming osteoblasts is enhanced by the presence of strontium as well as the rate of extracellular matrix formation and mineralization (new bone deposition) [4,5,7]. This effect is based on an influence of Sr2+ on the Wnt/b-catenin pathway resulting in enhanced extracellular matrix formation [4] and triggering of mitogenic signalling [8]. Furthermore, Bakker et al. recently found evidence that SrR affects the signalling between osteocytes and both osteoblasts and osteoclasts [11]. In vivo, a significant increase of bone mineral density associated with a decrease of osteoporosis-related vertebral and non-vertebral fracture risk was demonstrated in two phase 3 clinical trials under the administration of 2 g day1 strontium ranelate [12,13]. Still, the bioavailability of SrR is relatively low (20% [14]) and the actual concentration of strontium at a specific remodelling site cannot be measured and therefore remains unknown. Since highdose administration of strontium ranelate has sometimes been associated to osteomalacia in rats [15,16], a controlled, local release of divalent strontium ions might be preferable to the systemic administration in the treatment of osteoporotic bone defects. Therefore, strontium has been incorporated in apatite coatings of orthopedic or dental implants [17–19], or polymerbased bone cements [20]. Special attention has been given to

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.07.027

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calcium phosphate bone cements, which are frequently used in the treatment of bone defects and complicated fractures (also in osteoporotic bone). Calcium phosphate bone cements can integrate into the physiological bone remodelling process due to their solubility and bio-degradability and are therefore ideal to locally release Sr2+ into a defect of the bone [21]. Thus, the effect of Sr2+ incorporated into different bone cement formulations has been investigated in several studies [22–27]. However, most of these approaches involve the synthesis of Sr-containing calcium phosphate species and therefore require high temperature processing or elaborate precipitation techniques. In our work, we used two different routines to introduce up to 8.37 wt.% strontium into a well-characterized, hydroxyapatiteforming bone cement based on a-tricalcium phosphate [28]: either the addition of strontium carbonate (SrCO3, A-type modification) or the substitution of CaCO3 by SrCO3 (S-type) in the cement powder. While set A-type cements therefore are composed of a hydroxyapatite matrix filled with SrCO3 clusters, S-type cements are characterized by a homogeneous phase of Sr-substituted hydroxyapatite, as described recently in detail in a complementary study [29]. Here, we describe the positive influence of these novel strontium(II) modifications on in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs), the progenitors of osteoblasts which play a crucial role in bone regeneration. The release of Sr2+ from the modified cements was measured and correlated with proliferation and differentiation of hMSC cultured under the influence of different Sr2+ concentrations. Secondly, ionic interactions ([Sr2+], [Ca2+] and pH) between the cell culture medium and different cement variations as well as the effect of these variations on hMSC were studied. Finally, hMSCs were cultured in direct contact with the different cement types and characterized with biochemical and molecular biological methods regarding their proliferation and osteogenic differentiation.

2. Materials and methods 2.1. Sample preparation In this study, a hydroxyapatite-forming calcium phosphate cement (CPC) as first described by Khairoun et al. [28] was used as starting material and control. The CPC precursor consisted of 58 wt.% a-tricalcium phosphate (a-TCP, a-Ca3(PO4)2), 24 wt.% calcium hydrogen phosphate (monetite, CaHPO4), 8.5 wt.% calcium carbonate (CaCO3) and 8.5 wt.% hydroxyapatite (Ca10(PO4)6(OH)2) and was supplied by InnoTERE GmbH, Radebeul, Germany. During ageing in the presence of water, the precursor gradually sets into Ca-deficient hydroxyapatite, with a conversion ratio of 70% after 7 days. Four different strontium modifications were prepared as described previously in detail [29]. Briefly, in samples referred to as ‘‘A-type’’ strontium carbonate (SrCO3) was added in two concentrations (5 and 10 wt.%, labelled as A5 and A10, respectively). Upon setting, A-type cements form Ca-deficient hydroxyapatite matrix (comparable to CPC) with cluster-like SrCO3 agglomerates. Sam-

ples denoted as S50 and S100 were obtained by 50 and 100 wt.% substitution of CaCO3 by SrCO3 in the above-mentioned precursor formulation (see Table 1). By this ‘‘S-type’’ approach, a homogeneous substitution of Ca2+ by Sr2+ ions in the hydroxyapatite lattice of the set cement could be obtained, due to the formation of Srsubstituted hydroxyapatite, as has been shown recently [29]. Cement powder was mixed manually with 4 wt.% aqueous disodium hydrogen phosphate (Na2HPO4) solution using a powder-to-liquid ratio of 400 ll g1 to form a mouldable paste. Disc-shaped specimens designed to fit into standard 48-well tissue culture plates (10 mm diameter and 1 mm height) were formed using silicone moulds and cured for 4 days in water-saturated atmosphere in a sealed container at 37 °C, instead of immersion in water or aqueous solutions, which would lead to a partial release of strontium ions already during cement setting. Subsequently, samples were air dried and sterilized by c-radiation at 25 kGy. 2.2. Cement characterization 2.2.1. Cement surface characterization Cement surface was characterized prior to cell seeding by scanning electron microscopy (SEM, Phillips ESEM XL30, Eindhoven, The Netherlands) on samples coated with carbon (Leica EM SCD005, Leica Microsystems, Wetzlar, Germany). Furthermore, surface roughness was assessed on samples after sterilization as well as on samples immersed in basal cell culture medium (aMEM containing 9% FCS, 10 U ml1 penicillin, 100 lg ml1 streptomycin and 1% L-glutamine, all purchased from Biochrom, Berlin, Germany) for 3 days using white light interferometry (FRT MicroProf, CHR 150 N) and evaluated using the FRT Mark III software (V3.9.10, both Fries Research & Technology, Bergisch Gladbach, Germany). 2.2.2. Ion concentration measurement Ion release from the different cements as well as pH in the medium was investigated during immersion of set, disc-shaped cement samples in 1 ml basal cell culture medium (a-MEM containing 9% FCS, 10 U ml1 penicillin, 100 lg ml1 streptomycin and 1% L-glutamine, all purchased from Biochrom, Berlin, Germany) in semi-dynamic mode (regular complete medium change). A second set of samples was immersed in medium containing osteogenic supplements (OS+, see Section 2.3) and a third set of liquid samples was collected during cell culture (see Section 2.3.3). After 3, 5, 7, 14 and 21 days of incubation at 37 °C and 5% CO2, pH was measured (pH spear, Eutech Instruments, Nijkerk, The Netherlands), supernatants were completely removed from the sample and collected for ion quantification. Subsequently, 1 ml fresh medium was added to the cement samples. The supernatants were stored for subsequent analysis by inductively coupled plasma mass spectrometry (ICP-MS, IRIS Intrepid II XUV, Thermo Fisher Scientific, Waltham, USA). For analysis, samples were diluted in 13.5 ml water and 0.5 ml HNO3 (Carl Roth, Karlsruhe, Germany) and filtered using 0.45 lm filter (TPP, Trasadingen, Switzerland) to remove possible cement debris.

Table 1 Composition and relative Sr2+ content of strontium modified bone cement precursor powders. Label

CPC A5 A10 S50 S100

Description

Pure cement 5 wt.% SrCO3 addition 10 wt.% SrCO3 addition 50% substitution of CaCO3 by SrCO3 100% substitution of CaCO3 by SrCO3

Composition (wt.%)

Sr (wt.%)

a-TCP

DCPA

HA

CaCO3

SrCO3

58.00 55.80 53.20 58.00 58.00

24.00 23.10 22.00 24.00 24.00

8.50 8.20 7.80 8.50 8.50

8.50 8.20 7.80 4.25 0.00

0.00 4.80 9.20 4.25 8.50

0.00 2.83 5.40 4.28 8.37

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Sr2+ and Ca2+ ion concentrations were determined according to a calibration curve measured from element standards (High Purity, Charleston, USA). 2.3. Cell culture Primary human mesenchymal stem cells (hMSC), isolated from the bone marrow of three healthy male donors after obtaining informed consent and kindly provided by the Medical Clinic I, Dresden University Hospital ‘‘Carl Gustav Carus’’ (Prof. Martin Bornhäuser and co-workers), were used for cell culture experiments. The ethics commission of Technische Universität Dresden approved the application of hMSC for in vitro experiments. Cells were expanded in a-MEM containing 9% FCS, 10 U ml1 penicillin, 100 lg ml1 streptomycin and 1% L-glutamine (basal medium) at 37 °C and 5% CO2. Cells of the fourth passage were used for all cell culture experiments. To induce osteogenic differentiation of hMSC, basal medium was supplemented with 108 M dexamethasone, 5 mM b-glycerophosphate (-GP) and 0.05 mM ascorbic acid 2phosphate (all osteogenic supplements (OS) were purchased from Sigma–Aldrich, Taufkirchen, Germany) and all experiments were repeated with cells from three donors. 2.3.1. Effect of [Sr2+] on hMSC proliferation and osteogenic differentiation To study the effects of different Sr2+ concentrations on hMSC, 2  104 cells were seeded into 48-well tissue culture polystyrene (PS) plates and allowed to attach for 24 h in 350 ll basal medium. Subsequently, cells were cultivated in either basal or OS medium containing between 0.001 and 10 mM SrCl2 (Sigma–Aldrich) for 14 days with medium change every 3 to 4 days. After 1, 7 and 14 days, cell layers were washed with phosphate buffered saline (PBS) and stored at 80 °C until biochemical analysis of cell number and osteogenic differentiation. 2.3.2. Indirect cell culture The effects of ionic changes in the cell culture medium caused by differently modified calcium phosphate cements were studied using an indirect cell culture setup. 2  104 hMSCs were seeded into 48-well tissue culture PS plates and were allowed to attach for 24 h in basal medium. Simultaneously, cement samples were immersed in cell culture medium (basal and OS medium) and incubated for 24 h under cell culture conditions. From day 1 on, culture medium from the cements was collected and transferred onto the cells grown on PS every 3 to 4 days, while cements were immersed in fresh medium. Hence, cells were constantly exposed to medium ‘‘pre-conditioned’’ on cement samples. After 1, 7, 14 and 21 days, cell layers were washed with PBS and frozen at 80 °C until subsequent biochemical analysis. 2.3.3. Direct contact culture To study cell morphology, proliferation and osteogenic differentiation in direct contact with CPC and strontium(II)-modified cements, hMSCs were cultured directly on the cement samples. Prior to cell seeding, cement samples were soaked in 350 ll basal medium for 3 days to allow initial equilibration of the culture medium. 2  104 cells suspended in 150 ll basal medium were seeded onto each cement sample without removal of the equilibration medium, so the total volume after cell seeding was 500 ll. Tissue culture PS was used as additional reference. 24 h after seeding, the medium was changed to 350 ll, either basal or OS, and subsequently renewed every 3 to 4 days. Removed medium was collected and stored at 80 °C for ion concentration measurement. After 1, 7, 14 and 21 days, cell-seeded samples were washed with PBS and frozen at 80 °C until biochemical analysis. Moreover, 7 days after seeding one set of cell-seeded cement samples was

fixed for fluorescence microscopy (see Section 2.6). In order to increase RNA yield for qRT-PCR analysis of osteogenic differentiation, 4  104 cells suspended in 150 ll basal medium were seeded onto each cement sample and cultured under the same conditions until RNA isolation after 1, 14 and 21 days (see Section 2.5.2). 2.4. Cell proliferation hMSC proliferation was determined by means of intracellular lactate dehydrogenase (LDH) enzyme activity, which reflects the number of viable cells. Therefore, frozen samples were thawed and cell lysis was performed by incubation in 1% Triton X-100 (Sigma–Aldrich) for 50 min on ice and 10 min ultrasonic treatment. LDH activity was measured from aliquots of the cell lysates using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, USA) according to the manufacturer’s instructions and correlated with the cell number using a calibration line as described previously [30]. 2.5. Osteogenic differentiation Osteogenic differentiation of hMSC was characterized by biochemical quantification of alkaline phosphatase (ALP) enzyme activity as well as real time quantitative reverse transcription PCR (qRT-PCR) determination of mRNA levels coding for bone sialoprotein II (BSP II). 2.5.1. Colorimetric ALP activity assay ALP enzyme activity was measured in the cell lysates (see Section 2.4) using the pNpp-method: aliquots of the cell lysates were incubated with 1 mg ml1 p-nitrophenylphosphate (pNpp, Sigma– Aldrich) in 100 mM diethanolamine (pH 9.8) containing 1% Triton X-100 and 1 mM MgCl2 (Sigma–Aldrich) at 37 °C for 30 min. The reaction was stopped by 1 M NaOH and p-nitrophenolate concentration was measured spectrophotometrically at 405 nm (infinite M200 PRO, Tecan, Männedorf, Switzerland) and correlated to a calibration line measured from different dilutions of a 1 mM p-nitrophenol (pNp) stock solution to quantify the conversion of pNpp to pNp by ALP enzyme activity. Specific ALP activity (lmol pNp/ 30 min/106 cells) was then calculated with respect to the cell number in each sample as determined by intracellular LDH activity (see Section 2.4). 2.5.2. Quantitative real time PCR (qRT-PCR) Total RNA was isolated from cells cultivated on cement samples after 1, 7, 14 and 21 days of culture with the Quiagen RNeasy Mini Kit following the manufacturer’s instructions. Reverse transcription of RNA into cDNA was carried out using Superscript II Reverse Transcriptase (Invitrogen, Burlington, Canada) as described previously [31] and cDNA was used as a template for the real time PCR. Beside the respective cDNA, reaction mixes contained the TaqManÒ Gene Expression Assays (comprising specific primers and FAM labelled probes) and the TaqManÒ Fast Universal PCR Master Mix (both purchased from Life Technologies, Carlsbad, USA). Expression of the gene coding for the osteoblastic marker bone sialoprotein II (BSPII) as well as of the endogenous reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was quantified (Table 2). Real time PCR was performed using the Table 2 TaqManÒ gene expression assays used for gene expression analysis. Gene

Assay ID

RefSeq

Assay location

Amplicon length

BSPII GAPDH

Hs00173720_m1 Hs99999905_m1

NM_004967.3 NM_002046.4

283 229

95 122

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Fig. 1. SEM images of CPC and strontium-modified cement samples after 4 days of ageing in water-saturated atmosphere (left) as well as after 3 days of additional equilibration in cell culture medium (state prior to cell seeding, right).

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Applied Biosystems’ 7500 Real Time PCR Fast System (Life Technologies). After an initial cycle at 95 °C for 20 s, 40 cycles were run at 95 °C for 3 s and 60 °C for 30 s. Cycle threshold (Ct) values were used for relative quantification of gene expression by normalizing the expression of BSPII to GAPDH for each sample. The relative fold-change (related to the unmodified CPC on day 1) was calculated using the comparative Ct (2DDCt) method. 2.6. Cell morphology For fluorescence microscopy, cells cultured on different cement samples for 7 days were washed with PBS and fixed in 3.7 wt.% formaldehyde in PBS for 15 min at room temperature. Residual fixing solution was removed by washing with PBS and cells were permeabilized using 0.2 wt.% Triton X-100 in PBS for 3 min. Samples were washed with PBS and incubated with 1 wt.% bovine serum albumin (BSA) in PBS for 45 min to prevent non-specific binding of fluorescence dyes and minimize autofluorescence of the cement substrate. Cell nuclei and actin cytoskeleton staining was performed using 20 ng ml1 DAPI (40 ,6-diamidin-2-phenylindol, Invitrogen) and 25 ll ml1 AlexaFluor 488Ò phalloidin (Invitrogen) in 1 wt.% BSA in PBS for 45 min in the dark. Samples were washed in PBS again and imaged using a Leica TCS SP5 confocal scanning laser microscope (Leica Microsystems). 2.7. Statistics All cell culture experiments were repeated with cells obtained from three individual donors and representative results from one donor are shown. Measurements were performed in triplicates and results are expressed as mean ± standard deviation. Analysis of variance (ANOVA) for repeated measures was used to evaluate statistical significance P < 0.05 of the results and the Tukey method was used for post hoc analysis to determine multiple comparisons (Origin 8.5.0G, OriginLab, Northampton, USA). 3. Results 3.1. Cement characterization 3.1.1. Cement surface properties Biomaterial surface parameters such as microporosity and surface roughness are known to influence bone cell adhesion [32]. Therefore, cement surface roughness and morphology were investigated prior to cell seeding. In Fig. 1, SEM micrographs of cements aged for 4 days in water saturated atmosphere as well as after additional 3 days of equilibration in cell culture medium are shown. Cement samples consisted of precursor powder particles that were bound together by re-precipitated crystals, resulting in a highly structured, open porous surface structure. As confirmed by surface roughness analysis (Fig. 2), the grain size and therefore surface roughness slightly decreased for A- and S-type cement samples compared to CPC. This was probably due to the milling process during preparation of strontium-containing cement precursor powders and did not change during cement equilibration in cell culture medium, as can be seen from Fig. 2A and B. 3.1.2. Cement interactions with cell culture medium Ionic alterations of cell culture medium caused by the presence of calcium phosphate based biomaterials are known to influence the cellular response in vitro [33]. We therefore investigated the release of Sr2+ and the concentration of Ca2+ as well as the pH during cement immersion in basal and osteogenic cell culture medium. Upon immersion of cement samples in culture medium with or without osteogenic supplements, a colour change towards

Fig. 2. Surface roughness of CPC and strontium-modified cement samples after 4 days of ageing in water-saturated atmosphere (A) as well as after 3 days of additional equilibration in cell culture medium (state prior to cell seeding (B), as determined by white light interferometry).

yellow was observed, indicating a slight acidification. This acidification decreased for all materials except CPC within the first 3 to 5 days, whilst pH on CPC samples reached a minimum on day 7 (Fig. 3A). In Fig. 3B, the Ca2+ concentration in the supernatant as measured by ICP-MS is shown. Generally, the presence of cement resulted in a depletion of [Ca2+] compared to the reference (PS) independently of the presence of osteogenic supplements in the culture medium; however, the actual concentration varied over time. The lowest Ca2+ concentrations were found on CPC, whereas in the case of Sr-containing cements the depletion of Ca2+ was, after an initial phase, less intense, indicating a distinct difference in the ionic interactions of A- and S-type cements with the cell culture medium. Measurement of [Sr2+] in the supernatant of cements immersed in basal and osteogenic medium (Fig. 3C) showed that a higher strontium(II) content of the cement generally resulted in an enhanced Sr2+ release for both A- and S-type modifications. After an initial phase (until day 5), characterized by higher Sr2+ release, [Sr2+] in the supernatant decreased by 50% and almost constant concentrations were measured until day 21. No qualitative differences were found when comparing Sr2+ release under OS and OS+ conditions. However, despite the comparable release profiles, A- and S-type cements differed regarding the released ion concentrations. After 7 days, a balanced state of 0.029 and 0.036 mM for A5 and A10 samples, respectively, as well as 0.057 and 0.074 mM for S50 and S100, respectively, was found, revealing a generally higher release of strontium ions from S-type cements. Interestingly, no distinct differences were found when comparing the release rates (and also [Ca2+] and pH) in OS and OS+ medium samples collected during cell culture (see Supplementary Fig. S1). Altogether, cumulative Sr2+ release over 21 days did not exceed 0.54 wt.% (for A5 cements) of the total amount of Sr2+ incorporated in the respective cement. Therefore, Sr2+-release can be assumed to continue for longer time periods. 3.2. Influence of [Sr2+] on hMSC proliferation and osteogenic differentiation To study the effect of Sr2+ ion concentrations on proliferation and osteogenic differentiation of hMSC, up to 10 mM SrCl2 (and therefore well above those released from the cements investigated here) were added to the culture medium during a 14 day cell culture experiment (Fig. 4). Light microscopy during cell culture did not reveal changes in cell morphology for Sr-concentrations up to 5 mM, and no mineral precipitations were observed. Up to 0.1 mM, strontium(II) addition resulted in a slightly increased number of cells under osteogenic stimulation (OS+) after 21 days compared to strontium-free culture conditions (Fig. 4A). At strontium(II)

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Fig. 3. Alteration of pH (A), calcium(II) (B) and strontium(II) concentration (C) in the supernatant of modified calcium phosphate cements during 21 days of immersion under basal cell culture conditions (immersion in 1 ml aMEM containing 9% FCS at 37 °C, 5% CO2, left) as well as in OS+ medium (right) compared to cement-free control (Ref.).

Fig. 4. Impact of medium Sr2+ concentration on hMSC: proliferation of osteogenically induced (OS+) and non-induced (OS) hMSC over 14 days at different Sr2+ concentrations as determined by intracellular LDH activity (A and C). Osteogenic differentiation as determined by specific ALP activity measurement (B and D). Significant differences (p < 0.05) of selected samples compared to Sr2+-free medium are indicated (⁄).

concentrations of 1 mM and above, the number of living cells decreased drastically at later time points (14 and 21 days). Interestingly, the number of living cells was only slightly decreased at 1 mM SrCl2 in non-stimulated (OS) cells, suggesting a higher tolerance of non-stimulated hMSC towards elevated

Sr2+-concentrations (Fig. 4C). ALP enzyme activity was found to vary over time under the influence of different Sr2+ concentrations (Fig. 4B): whilst on day 14 a maximum was found at 0.1 mM, the highest activity after 21 days was found for 0.001 mM, suggesting a more advanced state of osteogenic differentiation under the

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influence of 0.1 mM Sr2+. No distinct ALP activity was found in OS cells (Fig. 4D). Hence, a mild stimulatory effect of Sr2+ concentrations up to 0.1 mM on proliferation and osteogenic differentiation of hMSC was demonstrated. 3.3. Indirect in vitro study To study the cumulative effect of ionic changes in the cell culture medium caused by the presence of CPC or strontium(II)-modified calcium phosphate cements on hMSC, cells were incubated with pre-conditioned medium. In Fig. 5, cell number (A, C) and specific ALP activity (B, D) of hMSC cultured in basal or OS medium incubated on different cements are shown. In all media preconditioned on strontium-containing cements, cells showed a significantly higher proliferation compared to the unmodified cement, although the highest proliferation was found in non-conditioned medium (referred to as control in Fig. 5). Interestingly, significantly more cells were observed if cultured in medium pre-conditioned on S-type compared to A-type cements in non-stimulated (OS) cultures only (Fig. 5C). This effect was most intense after 21 days. Specific ALP activity was also significantly increased in stimulated (OS+) cells cultured under the influence of medium preconditioned on Sr2+-containing cements compared to those cultured under the influence of CPC. Medium pre-conditioning on S-type-modified cements lead to higher ALP activity than on A-type materials. Still, the highest ALP was measured in unconditioned OS medium (Fig. 5B), while no increased ALP activity was found in OS cells (Fig. 5D). 3.4. Direct cell culture Cell adherence to CPC and Sr2+-modified calcium phosphate cements was quantified by cell number determination on day 1

(Table 3). While only 24.3% of cells initially seeded on CPC could be detected after 24 h, cell adherence was significantly enhanced on A- and, even more so, on S-type-modified cements (up to 60.1% on S100). Cell proliferation was assessed over 21 days through cell number determination carried out by measurement of intracellular LDH activity at different time points (Fig. 6A and C). Compared to CPC, where the cell number increased approximately 6- to 12-fold within 21 days under OS+ and OS conditions, respectively, proliferation was significantly increased on A- and, even more, on S-type strontium(II)-modified materials under both conditions. The highest proliferation was found on S100 cement samples with a cell density that had reached the 20-fold value within 21 days. Here, the cell density almost equalled the one measured on PS control. These data indicate that strontium(II) modification of the calcium phosphate cement used in this study (particularly S-type) leads to a significant improvement of cell adhesion and proliferation. In Fig. 6B and D, specific ALP activity of hMSC cultivated on CPC and modified cements over 21 days is shown. At all time points, ALP activity of OS+ cells was increased on strontium(II)-modified cements compared to CPC and in some cases almost equalled those found for OS+ cells cultured on PS. While no differences were found between cells cultured on A5 and A10 samples, significantly higher values were found in OS+ cells cultured on S100 compared to S50 cements after 14 as well as 21 days. Quantitative RT-PCR was used to further analyse the osteogenic differentiation of cells cultured directly on CPC and Sr2+-modified cements by quantifying the expression of the gene coding for BSPII, a late marker of osteogenic differentiation, after 7, 14 and 21 days of cultivation (Fig. 7). Strong impact of the strontium modification on expression of BSPII was observed. On day 7, a slight increase of mRNA coding for BSPII was detected only on S50 and the PS control under osteogenic stimulation. After 14 days, elevated transcript

Fig. 5. Indirect cell culture: proliferation (A and C) and differentiation (B and D) of osteogenically induced (OS+) and non-induced (OS) hMSC cultured over 21 days in medium pre-conditioned on CPC and strontium(II)-modified cements compared to unconditioned medium (control). Significant differences (p < 0.05) of selected samples compared to CPC are indicated (⁄).

Table 3 Relative amount (with respect to PS) of cells as determined 1 day after seeding of 2  104 cells per sample on CPC and strontium(II)-modified calcium phosphate cements (measured by intracellular LDH activity).

a

Sample

CPC

A5

A10

S50

S100

PS

Seeding efficiency (%)

24.3 ± 3.1

42.9 ± 2.9a

31.6 ± 4.8a

54.5 ± 5.1a

60.1 ± 10.6a

100

Significantly different from CPC (p < 0.05).

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Fig. 6. Direct cell culture: proliferation of osteogenically induced (OS+) and non-induced hMSC (OS) on CPC and different Sr2+-modified calcium phosphate cements over 21 days as determined by LDH activity measurement (A and C). Osteogenic differentiation as measured by specific ALP activity (B and D). Significant differences (p < 0.05) of selected samples compared to CPC are indicated (⁄).

the pure CPC; however, transcript levels were significantly higher in cells which were cultivated on strontium-modified cement samples. While on day 14 the cement type releasing the highest amount of Sr2+ (S100) seemed to induce the strongest BSPII expression, on day 21 the highest BSPII transcript level was detected for the modification with the lowest amount of strontium(II) (A5). Nearly no expression of BSPII was found in non-induced (OS) cells on all materials. Fig. 8 shows fluorescence micrographs of hMSC after cultivation for 7 days with osteogenic stimulation on CPC, A10 and S100 cement samples. Only a few cells were found on CPC samples, growing in clusters and exhibiting a rounded morphology, as can be seen from the cytoskeletal staining (actin/green fluorescence). On A10 samples, more cells had adhered on the surface and had a spread, osteoblast-like morphology, whilst on S100 cells formed a dense, partially multi-layered network that almost covered the entire sample surface. Fig. 7. Relative BSPII gene expression levels of osteogenically induced (OS+) and non-induced (OS) hMSC during 21 days of cultivation on CPC and different Sr2+modified calcium phosphate cements. Cells cultivated on PS were used as control. The relative gene expression of BSPII is represented as n-fold change in comparison to the unmodified CPC on the respective time point. Significant differences (p < 0.05) of the values measured on selected samples compared to CPC are marked as (⁄).

levels were measured in OS+ cells grown on all strontium-containing cements as well as on the PS control, but not on the unmodified CPC. On day 21, BSPII was also expressed by OS+ cells cultivated on

4. Discussion Strontium(II) ions have been demonstrated to both increase the bone formation by osteoblasts as well as to decrease the osteoclast-mediated resorption of the bone matrix [4,13]. Therefore, strontium has gained considerable attention in osteoporosis therapy because of its anti-resorptive and osteo-anabolic potential, which led to its clinical application in the form of strontium ranelate some years ago. Besides its systemic administration, a

Fig. 8. Fluorescence staining micrographs, demonstrating hMSC spreading and cytoskeletal organization after 7 days of culture on CPC and Sr2+-modified calcium phosphate cements under osteogenic stimulation. Confocal microscopic images (scale bar = 50 lm) with dual staining of cell nuclei (DAPI, blue) and actin filaments (phalloidin, green).

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local release of Sr2+ ions from bone cements used in the treatment of osteoporotic bone fractures and defects is considered a promising approach since it could help to avoid implications of high dose treatment and compensate for the relatively low bioavailability of the drug. In addition, the agent then is concentrated at the location of bone healing and can directly interact with all cell types present there. In this study, the influence of strontium(II) modification of a hydroxyapatite-forming bone cement on proliferation and osteogenic differentiation of hMSC was investigated in three steps. The impact of different Sr2+ ion concentrations in the cell culture medium, the effect of ionic changes in the medium caused by the presence of strontium-containing calcium phosphate cements as well as the cellular response in direct contact with the material were studied separately. Strontium was introduced into a well-defined calcium phosphate bone cement using two different preparation routes: either by the addition of SrCO3 or by partial or complete substitution of CaCO3 by SrCO3 in the same powder formulation [29]. In set cements, both modifications resulted in a slight reduction of the cement surface roughness (Figs. 1 and 2) that was merely altered during cement immersion in cell culture medium. Therefore it can be assumed that differences in cell attachment did not, or only to a small extent, depend on material-specific surface topography. The Sr2+ doses released from these modified cements under cell culture conditions depended not only on the absolute strontium content but also on the approach which was applied to introduce strontium into the cement (Fig. 3). Higher strontium content generally resulted in a higher release for both A- and S-type modification. For all modifications, the [Sr2+] measured in the supernatant was approximately two-times higher on day 3 compared to day 7, indicating an elevated initial release. Later on, the concentration remained almost constant – between 0.029 mM (A5) and 0.074 mM (S100) – despite of the regular medium change. As described recently, A- and S-type cements are characterized by the presence of SrCO3 clusters or the homogeneous partial substitution of Sr2+ in the apatite lattice, respectively [29]. Therefore, we assume that release of Sr2+ from S-type cements is a combination of dissolution of Sr-substituted apatite at the sample surface and diffusion-limited release from the bulk material, whilst in the case of A-type samples the dissolution of near-surface SrCO3 clusters characterizes the release profile. Interestingly, the release of Sr2+ was lower than previously described by us in a material characterization study when the same materials were aged in NaCl/Tris buffer solution [29]. We attribute this effect to the adsorption of proteins from the cell culture medium to the cement surface possibly acting as a diffusion barrier that would slow down ion release from the cement as well as interactions of proteins and Sr2+ in the solution. This adsorption could further vary between A- and S-type cement surfaces, which would in turn alter the release profiles, too. However, the released Sr2+ doses are within the magnitude of those reported to positively influence osteoblast-like cells in vitro (0.5–5 mM [5,34,35]). The cements therefore show a sustained release of strontium ions over at least 21 days, and, since the percentage Sr2+ release within 21 days did not exceed 0.54% of the amount in freshly prepared cements, a persisting release can be assumed for longer time periods. Still, these Sr2+ doses are low compared with strontium-releasing bone substitute materials described in the literature. For example, Landi et al. [36] prepared granules of 400–600 lm particle size from strontiumsubstituted HA (SrHA) that released 1.16 mg g1 Sr2+ within 1 week, whilst a strontium(II) substituted cement based on vaterite CaCO3/calcium hydrogen phosphate dihydrate has recently been described to release up to 8 mg g1 Sr2+ within only 1 day [24]. Here, the accumulated Sr2+ release of S100 accounted for only 0.013 mg g1. However, due to the different preparation routines,

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sample characteristics and experimental setups, these data are difficult to compare. According to the results of the Sr2+-release measurement, we investigated the impact of strontium concentrations of up to 10 mM on hMSC by adding different amounts of SrCl2 into basal and OS culture medium. Even for high [Sr2+] in both OS and OS+ media, no spontaneous mineral precipitation was observed. We found a maximum concentration 0.1 mM Sr2+, above which a deleterious effect of strontium(II) ions on proliferation and osteogenic differentiation has to be considered (Fig. 4). Concentrations up to 0.1 mM had a slight stimulating effect on these cellular processes. Interestingly, non-differentiated hMSC (cells cultured without osteogenic stimulation) appeared to tolerate slightly higher Sr2+ concentrations. Comparable differences in the sensitivity of cells of the osteoblast lineage and pluripotent mesenchymal cells have been described before [8]. The effect of Sr2+ on the osteogenic differentiation was described by Braux et al., who investigated primary human osteoblasts cultured in the presence of strontium(II) and found a maximum of osteogenic marker expression at Sr2+ concentrations of 5  105 M as well as an inhibitory effect of 103 M SrCl2 (and higher) on cell proliferation [34]. However, in other studies a stimulating effect of up to 2 mM SrCl2 on ALP expression and bone nodule formation in cultures of human umbilical-cord-derived mesenchymal stem cells (HUMSCs) as well as a positive influence of 1 mM Sr2+ on long-term cultures of MC3T3E1 osteoblast-like cells has been shown [4,5]. We conclude from our data that the strontium doses released from the modified cements used in this study (Fig. 3) were in a range that could promote proliferation as well as osteogenic differentiation of cells of the osteoblastic lineage and were not expected to have any deleterious effect on hMSCs. Indirect cell culture was performed to investigate the cumulative effects of strontium release and other ionic alterations of the medium caused by the presence of the cement, such as variations of pH, calcium and phosphate concentration, on hMSC. Typically, a slight acidification of culture medium as well as a decrease of medium [Ca2+] are characteristics of a-TCP-based CPCs in aqueous solutions [37] and can be attributed to the presence of calciumdeficient hydroxyapatite [38]. Here, medium acidification was observed particularly for CPC but also, to a smaller extent, for A- and S-type-modified cements (Fig. 3A). Calcium concentration in the cell culture medium decreased considerably on all cement types, with minimum concentrations of 0.26 mM on CPC after 21 days. Calcium(II) ion concentrations are known to have distinct influence on osteoblast proliferation and differentiation and a depletion of Ca2+ can inhibit both cellular processes [39,40]. In fact, cell number was reduced compared to standard medium when cells were treated with medium pre-conditioned on cements samples (Fig. 5). Still, this effect was less intense with medium conditioned on Srmodified cements, independently of the preparation routine. Therefore, the inhibiting effect of Ca2+ depletion on the proliferation of hMSC seems to be attenuated by the presence of strontium, either by the decreased uptake of Ca2+ from the medium in the presence of strontium in the cement or due to the direct effect of Sr2+ in the conditioned medium. In the same experiment, the highest ALP activity was also measured in cells treated with medium conditioned on strontium-containing cements. Again, possible explanations are the reduced Ca2+ depletion caused by the strontium modification as well as the presence of Sr2+ in the conditioned medium. However, it has to be considered that alterations in medium pH and [Ca2+] are highly affected by the chosen cell culture conditions (sample-to-medium ratio in particular) and therefore might not depict possible effects of the material in vivo correctly. In a third set of experiments, where cells were directly seeded onto the cements, initial cell adherence was low on pure CPC and cell number merely increased during the first week. This effect

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has been already described previously [41]. At this early stage, distinct cell spreading (Fig. 8) was only found in the presence of strontium(II), with cells covering almost the whole cement surface in a dense monolayer. A similar effect has been described by Xue et al. [42], who compared sintered SrHA with pure HA ceramic samples and found an improved attachment and higher proliferation of human osteoprecursor cells. In contrast to the unmodified CPC, increased cell adhesion and proliferation were found on the strontium(II)-modified cements that depended not only on the amount but also on the method of strontium modification (Fig. 6). Surface roughness, known to have a distinct influence on the cell attachment [32], only slightly decreased from CPC to A- and S-type cements and is therefore not considered to have caused these differences. It could rather be explained by the pH and ionic changes of the medium that were less intense in the case of Sr2+-modified cements. Interestingly, after 21 days, cell density of OS+ samples was much higher on S-typemodified cements compared to A-type materials while no clear difference between A- and S-type modifications was found in unstimulated samples. Still, these differences did not result in variations in Ca2+ or Sr2+ levels, suggesting that the cell layer did not cover the samples surface to an extent that would prevent ionic interaction (see Supplementary Fig. S1). Both cells cultured under OS+ and OS conditions were therefore exposed to ion concentrations similar to those measured for cell-free samples, independently of the respective cell density, confirming the hypothesis that osteoblastlike cells and undifferentiated hMSC are differently affected by the presence of Sr2+ions. Osteogenic differentiation seems to be stimulated by the general presence of strontium, which was shown by the enhanced levels of ALP enzyme activity on all Sr-modified cements. This is strengthened by the higher expression of the late marker of osteogenic differentiation, BSPII (Fig. 7). Only one study using primary human osteoprogenitor cells in direct contact with strontium-containing calcium phosphate bone cements has been published so far [24]. When cultured in medium that induces the osteogenic differentiation by dexamethasone, a stimulating effect of the strontium(II) modification on the proliferation could only be shown after 15 days and osteogenic differentiation (as described by expression of mRNA coding for osteogenic markers) was only affected by the presence of the cement in general. However, in contrast to our study these cements showed a high initial burst release of Sr2+ that was nevertheless excluded from the experimental setup since culture medium was refreshed prior to cell seeding. Other studies using cell lines to characterize strontium-containing brushite [22] and HA [43]-forming calcium phosphate cements confirmed the absence of cytotoxicity of the respective cements but found no systematic beneficial effect of the strontium modification. Only one study focusing on an a-tricalcium phosphate based, gelatine-containing cement describes a beneficial effect of strontium modification on the osteogenic differentiation of MG63 cells [44]. Altogether, our data suggest that a combined effect of reduced medium acidification and calcium depletion together with the release of bioactive Sr2+ from the modified cements (S-type in particular) caused the enhanced proliferation and osteogenic differentiation of hMSC on strontium-modified cements. 5. Conclusions In this study, the effect of strontium(II) released from a calcium phosphate bone cement on primary human mesenchymal stem cells has been studied. Two simple routines were used to prepare cements with strontium contents between 0.72 and 2.21 wt.% that sustainably release between 0.03 and 0.07 mM Sr2+ under in vitro conditions. Comparable strontium ion concentrations were shown

to enhance both the proliferation as well as the osteogenic differentiation of hMSC over 21 days, with an optimum in the range of 0.1 mM. However, the release rate of Sr2+ in cell culture medium differed from the release in NaCl buffer measured in a previous study [29]. This could be attributed to the presence of proteins and will be the subject of further experiments. Using an indirect as well as a direct contact cell culture setup we could demonstrate that the beneficial effect of strontium-modified cements is based on (I) the stimulatory effect of Sr2+ released in relevant doses from the cement and (II) the reduced tendency of SrCPC to alter cell culture medium pH and Ca2+ ion concentration. ALP enzyme activity as well as BSP II gene expression showed an enhanced osteogenic differentiation on Sr-containing materials. Altogether, cement formulations containing 8.5 wt.% SrCO3 (S100) had the most positive influence on hMSCs and are therefore considered as promising materials in the treatment of e.g. osteoporotic bone defects. A first animal trial in which the S100 cement was tested against the nonmodified CPC in a critical sized bone defect model in osteoporotic rats has confirmed the stimulatory effect of the strontium modification in vivo [45]. Acknowledgments The authors thank the German Research Society (DFG) for financial support and Prof. Dr. M. Bornhäuser and co-workers (Medical Clinic I, University Hospital Carl Gustav Carus Dresden) for providing the hMSC. We are grateful to Ms S. Brüggemeier (Centre for Translational Bone, Joint and Soft Tissue Research, Medical Faculty and University Hospital, Technische Universität Dresden) and Ms A. Voß (Leibniz-Institute for Solid State and Materials Research IFW Dresden) for excellent technical assistance and to B. Woltmann and Dr U. Hempel (Institute for Physiological Chemistry, TU Dresden) for fruitful discussions. This study was funded by the German Research Society (DFG) as part of the Collaborative Research Centre/Transregio 79 (SFB/TR 79 – subproject M2). Appendix A. Figure with essential colour discrimination Certain figures in this article, particularly Fig. 8, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio. 2013.07.027). Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2013. 07.027. References [1] Consensus development conference. Diagnosis, prophylaxis, and treatment of osteoporosis. Am J Med 1991;90:107–10. [2] Johnell JA, Kanis O. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int 2006;17:1726–33. [3] Rachner TD, Khosla S, Hofbauer LC. Osteoporosis: now and the future. Lancet 2011;377:1276–87. [4] Yang F, Yang D, Tu J, Zheng Q, Cai L, Wang L. Strontium enhances osteogenic differentiation of mesenchymal stem cells and in vivo bone formation by activating WNT/Catenin signaling. Stem Cells 2001;29:981–91. [5] Barbara A, Delannoy P, Denis BG, Marie PJ. Normal matrix mineralization induced by strontium ranelate in MC3T3-E1 osteogenic cells. Metabolism 2004;53:532–7. [6] Marie PJ. Optimizing bone metabolism in osteoporosis: insight into the pharmacologic profile of strontium ranelate. Osteoporos Int 2003;14:S9–12. [7] Bonnelye E, Chabadel A, Saltel F, Jurdic P. Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro. Bone 2008;42:129–38.

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