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A novel resorbable strontium-containing -calcium sulfate hemihydrate bone substitute: a preparation and preliminary study
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 045010 (http://iopscience.iop.org/1748-605X/9/4/045010) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 169.230.243.252 This content was downloaded on 26/08/2014 at 08:46
Please note that terms and conditions apply.
Biomedical Materials Biomed. Mater. 9 (2014) 045010 (13pp)
doi:10.1088/1748-6041/9/4/045010
A novel resorbable strontium-containing α-calcium sulfate hemihydrate bone substitute: a preparation and preliminary study Xue Li1,2,4, Chang-peng Xu2,4, Yi-long Hou1,4, Jin-qi Song1, Zhuang Cui1,2, Sheng-nan Wang1, Lei Huang1, Chang-ren Zhou3 and Bin Yu1,2 1
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China 2 Key Laboratory of Bone and Cartilage Regenerative Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China 3 Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632, People’s Republic of China E-mail: [email protected] Received 22 December 2013, revised 3 June 2014 Accepted for publication 5 June 2014 Published 16 July 2014 Abstract
Distraction osteogenesis after aggrieved bone segment resections is promising in the treatment of bone tumors and osteomyelitis. However, there is ambiguity with regard to the optimal choice of bone substitute, with biodegradability and excellent bone repair performance constituting key requirements. The purpose of this study was to develop a novel resorbable strontium-containing α-calcium sulfate hemihydrate (Sr-CaS) bone substitute to provide an alternative option for surgeons that better meets these requirements. The Sr-CaS was prepared using co-precipitation and hydrothermal methods and analyzed using x-ray diffraction (XRD), Fourier transform infrared (FTIR) scanning and thermogravimetric differential scanning calorimeter (TG–DSC) patterns. Cytotoxicity by tetrazolium bromide (MTT), sub-acute toxicity and hemolysis tests were performed to assess the initial biocompatibility of the new bone substitute. Radiographic analysis, micro-CT measurements and histological observation were used to evaluate the bone repair ability in rat tibia bone defects. The XRD and FTIR patterns of Sr-CaS were both very similar to CaS and the product had comparable characteristics similar to α-CaS as demonstrated by TG-DSC. Cytotoxicity of the substitute was class 1 (no cytotoxicity) and hemolysis was 4.3% (no hemolysis). Sub-acute toxicity was not seen after a 14 day evaluation. The substitute was radio-opaque. The empty group exhibited the lowest levels of both bone mineral densities (BMD) and bone volume/total volume (BV/TV) of the defects when compared to all other groups. The two Sr-CaS groups resulted in significantly greater BMDs and BV/TV of the defect compared to the CaS only group. However, there was no significant difference between the 5% and 10% Sr-CaS groups. The Sr-CaS was resorbable with satisfactory biocompatibility. The doped strontium ions enhanced the bone repair performance of CaS in a rat model and the new substitute demonstrated promising results for clinical use. Keywords: strontium-containing α-calcium sulfate hemihydrate, resorbable bone substitute, biocompatibility, bone repair (Some figures may appear in colour only in the online journal)
4
Each author contributed equally to this study.
1748-6041/14/045010+13$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
X Li et al
Biomed. Mater. 9 (2014) 045010
1. Introduction
observed in soft tissue surrounding strontium-substituted bioactive glass (BG-Sr). In addition, the presence of Sr was verified to have few effects on unstimulated cells, but it decreased the production of pro-inflammatory cytokines and the chemokine interleukin 8 in LPS-stimulated cell-conditioned medium, so strontium may be involved in the control of inflammatory processes following calcium phosphate (BCP) phagocytosis by human monocytes [20]. Therefore, we further hypothesized that strontium-containing α-calcium sulfate (Sr-CaS) would have better bone repair performance than the simple CaS in theory. The purpose of this study was to test the feasibility of integrating strontium into α-calcium sulfate hemihydrates to form a new type of biodegradable bone substitute, and to test whether this novel material will display the combined merits of strontium and CaS in the treatment of bone cavity defects. The present study determined the biocompatibility of the novel composite, strontium-containing α-calcium sulfate hemihydrates (Sr-CaS), in both the cells and mouse models, and its excellent bone repair performance in rat models.
As calcium sulfate (CaS) has excellent biodegradability and biocompatibility [1–3], it is often used as a spacer for bone defects to prevent the ingrowth of fibrous tissue and to facilitate bone ingrowth [4, 5]. In distraction osteogenesis using an external fixator, it could be used after the whole aggrieved bone segment resection for the treatment of bone tumors and osteomyelitis to preserve the limb length [6]. CaS works well to increase the time for distraction osteogenesis with synchronized biodegradation. Medical grade CaS is known as α-calcium sulfate hemihydrate, which is identical to β-calcium sulfate hemihydrate in chemical characteristics but has different physical properties. The former is characterized with rod- and prism-shaped crystals, while the latter is characterized with the aggregation of irregular crystals and interstitial capillary pores [7]. Although the two forms of hemihydrate will both translate into dihydrate when mixed with water, the α-hemihydrate transformer is stronger, less soluble and superior in density compared to the β-hemihydrate variant [8]. Therefore, the CaS referred to in medical literature is usually α-calcium sulfate hemihydrates. Excellent biodegradability and biocompatibility of CaS make it an outstanding bone substitute with properties that are more similar to the normal bone than any other substitute discovered thus far. It is well known that CaS is an undoubted osteoconductive matrix that facilitated the invasion of capillaries and perivascular mesenchymal tissue, as well as osteoprogenitor cells. CaS has also been reported to be capable of enhancing bone formation, with results comparable with autogenous bone [9]. Furthermore, CaS has been shown to promote vascularization [10]. However, of more clinical interest is how to improve the bone repair performance of CaS to accelerate bone healing. We hypothesized that if CaS was integrated with a factor that had a confirmed osteoinductive effect, the novel composite might exhibit better bone repair performance than the simple CaS alone. Strontium has been shown to increase bone formation and reduce bone resorption [11, 12] In addition, divalent strontium has been reported to benefit bone collagen synthesis in vitro [13]. Thus, Sr-doped materials were widely studied in bone tissue engineering. In an in vivo study, strontium-containing hydroxyapatite (Sr-HA) was shown to accelerate bone growth and osteointegration [14]. Zhang et al [15]. demonstrated that strontium-containing mesoporous bioactive glass (Sr-MBG) fabricated using a three-dimensional (3D) printing technique was of good bone forming bioactivity, controlled ion release and enhanced mechanical strength, so has potential application in bone regeneration. Several studies reported that strontiumsubstituted bioactive glass (SrBG) exhibited better ability to promote osteogenesis than the bioactive glass without strontium [16, 17]. Consistent with previous studies, Sr-substituted calcium silicate (SrCS) ceramic scafolds combining the functions of Sr and Si elements were developed with the goal of promoting osteoporotic bone defect repair [18]. Furthermore, despite the ability of osteogenesis, some other effects of Sr have also been proven. Jebahi et al [19] revealed that the protective action against reactive oxygen species (ROS) was clearly
2. Material and methods 2.1. Materials 2.1.1. Preparation of Sr-CaSO4·2H2O. Sr-CaSO4·2H2O pow-
der was prepared using a co-precipitation method according to the following chemical formulas: Ca(OH)2 + H2SO4 = CaSO4 ↓ + 2H2O Sr(OH)2 + H2SO4 = SrSO4 ↓ + 2H2O.
Theoretically, the mole ratio of (Ca+Sr)/S was 1:1 and the mole ratio of Sr/Ca was 1:9. First, 0.05 mol Sr(OH)2 ·8H2O and 0.45 mol Ca(OH)2 were poured into 300 ml deionized water. Then, 0.5 mol H2SO4 was diluted to 50 ml which was added dropwise to the basic suspension liquid while being stirred at 30 °C. The pH value of the slurry across the whole reaction process was held at 8.5 by pH electrode before H2SO4 was finished. The reaction mixture was stirred for 3 h. After incubation, the slurry was filtered through filter paper and dried at 50 °C for 4 h in a stove. Finally, the resulting gypsum was ground into a powder. 2.1.2. Preparation of Sr-α-CaSO4·0.5H2O. A hydrothermal
reaction was used to prepare Sr-α-CaSO4·0.5H2O. The principle is: °c CaSO4 ·2 H2O 130 ⎯⎯⎯⎯⎯⎯→ CaSO4 ·0.5H2 O+ 1.5 H2O.
The elementary product was transferred into fine powder by a planetary ball mill and 200 mesh. Then, the semi- finished mixture was heated in a Muffle furnace (F46240CM, Thermolyne, USA) at a 15% mass ratio of product/deionized water at 130 °C for 6 h. The slurry was poured after a few minutes while hot and subsequently filtered subsequently. The product was stoved at 50 °C for 4 h and pulverized again. The powder samples were examined by x-ray diffraction (XRD, D/Max-2550 pc, Rigaku Inc.) with a voltage of 40 kV 2
X Li et al
Biomed. Mater. 9 (2014) 045010 −1
and a current of 40 mA at a scanning rate of 0.75° s in the 2 range from 5° to 80°. Fourier transform infrared (FTIR) spectra were obtained using a FTIR spectrometer (FTS-7, Bio-Rad, USA) and recorded from 4000 to 400 cm. The thermogravimetric differential scanning calorimeter (TG–DSC) patterns were obtained with differential scanning calorimeter (DIAMOND DSC, PE USA) to determine the strontium-containing calcium sulfate phases and crystal water content. A 20 mg sample was sealed in an Al2O3 crucible with a lid and scanned at a rate of 10 °C min−1 under N2 gas atmosphere. 2.2. Biological compatibility evaluation
Biological evaluation was conducted in accordance with the International Standards Organization (ISO) 10993-1-2009. All experimental protocols were approved by the Institutional Animal Ethics Committee. 2.2.1. Extracts from Sr-CaS cement. The prepared powder and deionized water were mixed at a concentration of 3 g ml−1 ratio in the shape of cylindrical specimens of 5 mm in diameter and 1 cm in height. The setting time for the mixing is 30 min or so. Each specimen was subsequently sterilized by 60 Co irradiation before use. The material was extracted after being immersed in normal saline at a ratio of 3 cm2 ml−1 for 72 h at 37 °C in accordance with ISO 10993-12-2009 [21]. 2.2.2. Cytotoxicity. L929 cells, a preferred established mouse fibroblast cell line according to ISO 10993-5-2009 [21], were cultured in a standard culture medium containing 10% fetal bovine serum (FBS), 200 mg ml−1 penicillin, and 200 mg ml−1 streptomycin. Once 1 × 105 ml−1 cell suspension was achieved, the three 96-well microtitre plates were inoculated with prepared cells, 100 µl per well. Then the plates were placed in a CO2 incubator with a humidified atmosphere and incubated for 24 h at 37 °C. The next 36 wells on each plate were inoculated with 6 different contents and thus divided into 6 groups of volume 100 µl: fresh culture medium (negative control), 0.64% phenol solution (positive control), 100% extract, 75% extract, 50% extract and 25% extract. Subsequently, the cells were incubated for another 72 h. Tetrazolium bromide (MTT) assay was performed in the six wells of each group on the three plates, respectively, at 24, 48 and 72 h. In all the wells, the previous medium was exchanged with 20 µl colorimetric reagent MTT (5 mg ml−1) at each time point. Incubation was terminated after MTT staining for another 4 h. The microtitre plates were shaken for 10 min after 150 µl DMSO was added into each well. Then, optical density of the medium was measured at 490 nm by a plate reader (Biotek, USA). Finally, RGR (relative growth rate) values and classification of cytotoxicity were calculated according to ISO 10993-5-2009 [22].
Figure 1. X-ray diffraction pattern (a), Fourier transform infrared spectrum (b) and differential scanning calorimeter pattern (c) of strontium-containing calcium sulfate (Sr-CaS) powder. In the DSC pattern, A represented the TG change and B was the DSC line. Table 1. Results of the MTT assay after 72 h culture.
Group
OD (Mean ± SD)
RGR (%) Cytotoxicity
Negative control 25% extract 50% extract 75% extract 100% extract Positive control
0.555 ± 0.024 0.530 ± 0.039 0.542 ± 0.044 0.533 ± 0.026 0.542 ± 0.033 0.080 ± 0.005
100 95.5 97.7 96.0 97.7 14.4
0 I I I I IV
dose of 50 ml kg−1 abiding by the maximum dosage criteria of ISO 10993-11-2009 [23], while normal saline was injected in the control group. Injections were conducted daily in both groups for 2 weeks. The injection syringe had specifications: volume capacity 2 ml, and accuracy 0.1 ml. The results were
2.2.3. Subacute toxicity. Twenty mice (Guangzhou, Guangdong, China) were randomized into an experiment group and a control group, 50% male in each group. The extract solution was injected in the experiment group at an intraperitoneal 3
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Biomed. Mater. 9 (2014) 045010
Figure 2. MTT assays for cell proliferation of L-929 cell cultured in different composite groups for 1, 2 and 3 days.
evaluated based on the common clinical signs and observations as specified in ISO 10993-11-2009 [23]. Animal body weights were also recorded pre-experiment, and at 1 and 2 weeks postexperiment. Furthermore, clinical pathology, organ weights, and histopathology were evaluated at 2 weeks post-experiment.
with physiological saline to remove any residual bone and bone marrow. The 80 legs of the 40 rats were randomized into 4 even groups (20 legs in each group), the defects in these 4 groups were filled with different materials: group A (nothing for empty control), group B (CaS), group C (5% Sr-CaS) and group D (10% Sr-CaS). The defects were filled flush to the anterior cortex manually with paste-form material before being allowed to set in situ. After surgery, the skin was carefully sutured and injected with antibiotics (3% Penicillin). All mice recovered well from surgery, and were housed separately in plastic cages for 12 weeks. Food and water were supplied ad libitum. Digital radiographs were taken immediately, 4, 8 and 12 weeks post-operation under anesthesia using an Oralix AC Densomat x-ray machine (Gendex Dental System, Milan, Italy). The tibias were harvested at 4, 8 and 12 weeks postoperatively for histological and micro-computed tomography evaluations.
2.2.4. Hemolysis test. The original extracts and diluted anticoagulated fresh rabbit blood were used in the test. According to the hemolysis test protocol, the test was performed in three groups: rabbit blood plus the extract in experimental group, rabbit blood plus distilled water as a positive control and rabbit blood plus physiological saline as a negative control. After 0.2 ml rabbit blood was gradually added into the extract, the centrifuge tube was placed for water bath for 1 h at 37 °C. Subsequently, 5 min centrifugation was performed at 3000 revolutions s−1. After that, the OD value at 540 nm of the supernatant was determined by a spectrometer. The rate of hemolysis was finally calculated as described in ISO 109934-2009 [24].
2.3.2. Micro-computed tomography. 20 rats (40 legs) euthanized at 12 weeks postoperatively were used for micro-computed tomography. The morphology of the reconstructed tibial cortex was assessed using an animal micro-CT scanner (eXplore Locus, GE Healthcare Biosciences, London, UK). Briefly, the specimens were scanned with a 55 kVp energy setting, intensity of 145 mA with 200 ms acquisition time and no frame average in high-resolution mode which provided a voxel resolution of 12 µm. After micro-CT scan, the defect region was identified by a contour as a traced region of interest (ROI), the relative measurements of which were calculated, including bone mineral densities (BMDs) and bone volume/ total volume (BV/TV).
2.3. Implantation in rats 2.3.1. Bone defect animal experiments. Forty-eight week-
old male Wistar rats weighing 200–250 g were used to create bone defect models as described previously [25, 26]. The general anesthesia was induced by intraperitoneal injection of Nembutal at 40 mg kg−1 body weight. The skin over the proximal tibia was incised and the periosteum was cleared using a periosteal elevator. A defect 3 mm wide and 5 mm long was created using a micro-burr with a 2.5 mm tip, starting 10 mm below the articular surface in the anteromedial cortex at both tibias. The defects and intramedullary canals were flushed 4
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Biomed. Mater. 9 (2014) 045010
Figure 3. The changes regarding body weight (a), hematological indexes ((b)–(e)) or organ weight ((f) and (g)) between the experiment and control groups. HCT, hematokrit (L L−1); MCV, mean corpuscular volume (fl); PLT, platelet count (×1010 L−1); HGB, hemoglobin (×10 g L−1); RBC, red blood cell (×1012 L−1); WBC, white blood cell, (×109 L−1); BUN, blood urea nitrogen (mmol L−1); CHO, total cholesterol (mmol L−1); GLU, glucose (mmol L−1); TG, triglyceride (mmol L−1); ALT, alanine aminotransferase (U L−1); AST, aspartate amino transferase (×10 U L−1); CRE, creatinine (µmol L−1); TP, total protein (g L−1); body weight and organ weight were all measured in grams.
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Biomed. Mater. 9 (2014) 045010
Figure 4. Fourteen days after the first injection: no inflammation or necrotic tissue was observed in the histological analysis of the liver ((a) and (b)), heart ((c) and (d)), spleen ((e) and (f)), lung ((g) and (h)) and kidney ((i) and (j)) The left column was the control group and the right was the experiment group (hematoxylin and eosin [H&E] stain, × 100 magnification). 6
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Biomed. Mater. 9 (2014) 045010
Table 2. Results of hemolysis test.
Group
OD (Mean ± SD)
Sr-CaS Negative group Positive group
0.050 ± 0.002 0.023 ± 0.001 0.656 ± 0.012
3.2. Biocompatibility test Haemolysis rate (%)
3.2.1. Cytotoxicity. The cell viability of the extracted solution
of the Sr-CaS composite measured by MTT assay at 1, 2 and 3 days after cell culture is shown in figure 2. There were no significant differences regarding the growth rate among the experimental and negative control groups (P > 0.05). Not surprisingly, the positive control group presented the least cell viability which was significantly lower than that in all the other groups (P
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My IOPscience
A novel resorbable strontium-containing -calcium sulfate hemihydrate bone substitute: a preparation and preliminary study
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 045010 (http://iopscience.iop.org/1748-605X/9/4/045010) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 169.230.243.252 This content was downloaded on 26/08/2014 at 08:46
Please note that terms and conditions apply.
Biomedical Materials Biomed. Mater. 9 (2014) 045010 (13pp)
doi:10.1088/1748-6041/9/4/045010
A novel resorbable strontium-containing α-calcium sulfate hemihydrate bone substitute: a preparation and preliminary study Xue Li1,2,4, Chang-peng Xu2,4, Yi-long Hou1,4, Jin-qi Song1, Zhuang Cui1,2, Sheng-nan Wang1, Lei Huang1, Chang-ren Zhou3 and Bin Yu1,2 1
Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China 2 Key Laboratory of Bone and Cartilage Regenerative Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, People’s Republic of China 3 Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632, People’s Republic of China E-mail: [email protected] Received 22 December 2013, revised 3 June 2014 Accepted for publication 5 June 2014 Published 16 July 2014 Abstract
Distraction osteogenesis after aggrieved bone segment resections is promising in the treatment of bone tumors and osteomyelitis. However, there is ambiguity with regard to the optimal choice of bone substitute, with biodegradability and excellent bone repair performance constituting key requirements. The purpose of this study was to develop a novel resorbable strontium-containing α-calcium sulfate hemihydrate (Sr-CaS) bone substitute to provide an alternative option for surgeons that better meets these requirements. The Sr-CaS was prepared using co-precipitation and hydrothermal methods and analyzed using x-ray diffraction (XRD), Fourier transform infrared (FTIR) scanning and thermogravimetric differential scanning calorimeter (TG–DSC) patterns. Cytotoxicity by tetrazolium bromide (MTT), sub-acute toxicity and hemolysis tests were performed to assess the initial biocompatibility of the new bone substitute. Radiographic analysis, micro-CT measurements and histological observation were used to evaluate the bone repair ability in rat tibia bone defects. The XRD and FTIR patterns of Sr-CaS were both very similar to CaS and the product had comparable characteristics similar to α-CaS as demonstrated by TG-DSC. Cytotoxicity of the substitute was class 1 (no cytotoxicity) and hemolysis was 4.3% (no hemolysis). Sub-acute toxicity was not seen after a 14 day evaluation. The substitute was radio-opaque. The empty group exhibited the lowest levels of both bone mineral densities (BMD) and bone volume/total volume (BV/TV) of the defects when compared to all other groups. The two Sr-CaS groups resulted in significantly greater BMDs and BV/TV of the defect compared to the CaS only group. However, there was no significant difference between the 5% and 10% Sr-CaS groups. The Sr-CaS was resorbable with satisfactory biocompatibility. The doped strontium ions enhanced the bone repair performance of CaS in a rat model and the new substitute demonstrated promising results for clinical use. Keywords: strontium-containing α-calcium sulfate hemihydrate, resorbable bone substitute, biocompatibility, bone repair (Some figures may appear in colour only in the online journal)
4
Each author contributed equally to this study.
1748-6041/14/045010+13$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
X Li et al
Biomed. Mater. 9 (2014) 045010
1. Introduction
observed in soft tissue surrounding strontium-substituted bioactive glass (BG-Sr). In addition, the presence of Sr was verified to have few effects on unstimulated cells, but it decreased the production of pro-inflammatory cytokines and the chemokine interleukin 8 in LPS-stimulated cell-conditioned medium, so strontium may be involved in the control of inflammatory processes following calcium phosphate (BCP) phagocytosis by human monocytes [20]. Therefore, we further hypothesized that strontium-containing α-calcium sulfate (Sr-CaS) would have better bone repair performance than the simple CaS in theory. The purpose of this study was to test the feasibility of integrating strontium into α-calcium sulfate hemihydrates to form a new type of biodegradable bone substitute, and to test whether this novel material will display the combined merits of strontium and CaS in the treatment of bone cavity defects. The present study determined the biocompatibility of the novel composite, strontium-containing α-calcium sulfate hemihydrates (Sr-CaS), in both the cells and mouse models, and its excellent bone repair performance in rat models.
As calcium sulfate (CaS) has excellent biodegradability and biocompatibility [1–3], it is often used as a spacer for bone defects to prevent the ingrowth of fibrous tissue and to facilitate bone ingrowth [4, 5]. In distraction osteogenesis using an external fixator, it could be used after the whole aggrieved bone segment resection for the treatment of bone tumors and osteomyelitis to preserve the limb length [6]. CaS works well to increase the time for distraction osteogenesis with synchronized biodegradation. Medical grade CaS is known as α-calcium sulfate hemihydrate, which is identical to β-calcium sulfate hemihydrate in chemical characteristics but has different physical properties. The former is characterized with rod- and prism-shaped crystals, while the latter is characterized with the aggregation of irregular crystals and interstitial capillary pores [7]. Although the two forms of hemihydrate will both translate into dihydrate when mixed with water, the α-hemihydrate transformer is stronger, less soluble and superior in density compared to the β-hemihydrate variant [8]. Therefore, the CaS referred to in medical literature is usually α-calcium sulfate hemihydrates. Excellent biodegradability and biocompatibility of CaS make it an outstanding bone substitute with properties that are more similar to the normal bone than any other substitute discovered thus far. It is well known that CaS is an undoubted osteoconductive matrix that facilitated the invasion of capillaries and perivascular mesenchymal tissue, as well as osteoprogenitor cells. CaS has also been reported to be capable of enhancing bone formation, with results comparable with autogenous bone [9]. Furthermore, CaS has been shown to promote vascularization [10]. However, of more clinical interest is how to improve the bone repair performance of CaS to accelerate bone healing. We hypothesized that if CaS was integrated with a factor that had a confirmed osteoinductive effect, the novel composite might exhibit better bone repair performance than the simple CaS alone. Strontium has been shown to increase bone formation and reduce bone resorption [11, 12] In addition, divalent strontium has been reported to benefit bone collagen synthesis in vitro [13]. Thus, Sr-doped materials were widely studied in bone tissue engineering. In an in vivo study, strontium-containing hydroxyapatite (Sr-HA) was shown to accelerate bone growth and osteointegration [14]. Zhang et al [15]. demonstrated that strontium-containing mesoporous bioactive glass (Sr-MBG) fabricated using a three-dimensional (3D) printing technique was of good bone forming bioactivity, controlled ion release and enhanced mechanical strength, so has potential application in bone regeneration. Several studies reported that strontiumsubstituted bioactive glass (SrBG) exhibited better ability to promote osteogenesis than the bioactive glass without strontium [16, 17]. Consistent with previous studies, Sr-substituted calcium silicate (SrCS) ceramic scafolds combining the functions of Sr and Si elements were developed with the goal of promoting osteoporotic bone defect repair [18]. Furthermore, despite the ability of osteogenesis, some other effects of Sr have also been proven. Jebahi et al [19] revealed that the protective action against reactive oxygen species (ROS) was clearly
2. Material and methods 2.1. Materials 2.1.1. Preparation of Sr-CaSO4·2H2O. Sr-CaSO4·2H2O pow-
der was prepared using a co-precipitation method according to the following chemical formulas: Ca(OH)2 + H2SO4 = CaSO4 ↓ + 2H2O Sr(OH)2 + H2SO4 = SrSO4 ↓ + 2H2O.
Theoretically, the mole ratio of (Ca+Sr)/S was 1:1 and the mole ratio of Sr/Ca was 1:9. First, 0.05 mol Sr(OH)2 ·8H2O and 0.45 mol Ca(OH)2 were poured into 300 ml deionized water. Then, 0.5 mol H2SO4 was diluted to 50 ml which was added dropwise to the basic suspension liquid while being stirred at 30 °C. The pH value of the slurry across the whole reaction process was held at 8.5 by pH electrode before H2SO4 was finished. The reaction mixture was stirred for 3 h. After incubation, the slurry was filtered through filter paper and dried at 50 °C for 4 h in a stove. Finally, the resulting gypsum was ground into a powder. 2.1.2. Preparation of Sr-α-CaSO4·0.5H2O. A hydrothermal
reaction was used to prepare Sr-α-CaSO4·0.5H2O. The principle is: °c CaSO4 ·2 H2O 130 ⎯⎯⎯⎯⎯⎯→ CaSO4 ·0.5H2 O+ 1.5 H2O.
The elementary product was transferred into fine powder by a planetary ball mill and 200 mesh. Then, the semi- finished mixture was heated in a Muffle furnace (F46240CM, Thermolyne, USA) at a 15% mass ratio of product/deionized water at 130 °C for 6 h. The slurry was poured after a few minutes while hot and subsequently filtered subsequently. The product was stoved at 50 °C for 4 h and pulverized again. The powder samples were examined by x-ray diffraction (XRD, D/Max-2550 pc, Rigaku Inc.) with a voltage of 40 kV 2
X Li et al
Biomed. Mater. 9 (2014) 045010 −1
and a current of 40 mA at a scanning rate of 0.75° s in the 2 range from 5° to 80°. Fourier transform infrared (FTIR) spectra were obtained using a FTIR spectrometer (FTS-7, Bio-Rad, USA) and recorded from 4000 to 400 cm. The thermogravimetric differential scanning calorimeter (TG–DSC) patterns were obtained with differential scanning calorimeter (DIAMOND DSC, PE USA) to determine the strontium-containing calcium sulfate phases and crystal water content. A 20 mg sample was sealed in an Al2O3 crucible with a lid and scanned at a rate of 10 °C min−1 under N2 gas atmosphere. 2.2. Biological compatibility evaluation
Biological evaluation was conducted in accordance with the International Standards Organization (ISO) 10993-1-2009. All experimental protocols were approved by the Institutional Animal Ethics Committee. 2.2.1. Extracts from Sr-CaS cement. The prepared powder and deionized water were mixed at a concentration of 3 g ml−1 ratio in the shape of cylindrical specimens of 5 mm in diameter and 1 cm in height. The setting time for the mixing is 30 min or so. Each specimen was subsequently sterilized by 60 Co irradiation before use. The material was extracted after being immersed in normal saline at a ratio of 3 cm2 ml−1 for 72 h at 37 °C in accordance with ISO 10993-12-2009 [21]. 2.2.2. Cytotoxicity. L929 cells, a preferred established mouse fibroblast cell line according to ISO 10993-5-2009 [21], were cultured in a standard culture medium containing 10% fetal bovine serum (FBS), 200 mg ml−1 penicillin, and 200 mg ml−1 streptomycin. Once 1 × 105 ml−1 cell suspension was achieved, the three 96-well microtitre plates were inoculated with prepared cells, 100 µl per well. Then the plates were placed in a CO2 incubator with a humidified atmosphere and incubated for 24 h at 37 °C. The next 36 wells on each plate were inoculated with 6 different contents and thus divided into 6 groups of volume 100 µl: fresh culture medium (negative control), 0.64% phenol solution (positive control), 100% extract, 75% extract, 50% extract and 25% extract. Subsequently, the cells were incubated for another 72 h. Tetrazolium bromide (MTT) assay was performed in the six wells of each group on the three plates, respectively, at 24, 48 and 72 h. In all the wells, the previous medium was exchanged with 20 µl colorimetric reagent MTT (5 mg ml−1) at each time point. Incubation was terminated after MTT staining for another 4 h. The microtitre plates were shaken for 10 min after 150 µl DMSO was added into each well. Then, optical density of the medium was measured at 490 nm by a plate reader (Biotek, USA). Finally, RGR (relative growth rate) values and classification of cytotoxicity were calculated according to ISO 10993-5-2009 [22].
Figure 1. X-ray diffraction pattern (a), Fourier transform infrared spectrum (b) and differential scanning calorimeter pattern (c) of strontium-containing calcium sulfate (Sr-CaS) powder. In the DSC pattern, A represented the TG change and B was the DSC line. Table 1. Results of the MTT assay after 72 h culture.
Group
OD (Mean ± SD)
RGR (%) Cytotoxicity
Negative control 25% extract 50% extract 75% extract 100% extract Positive control
0.555 ± 0.024 0.530 ± 0.039 0.542 ± 0.044 0.533 ± 0.026 0.542 ± 0.033 0.080 ± 0.005
100 95.5 97.7 96.0 97.7 14.4
0 I I I I IV
dose of 50 ml kg−1 abiding by the maximum dosage criteria of ISO 10993-11-2009 [23], while normal saline was injected in the control group. Injections were conducted daily in both groups for 2 weeks. The injection syringe had specifications: volume capacity 2 ml, and accuracy 0.1 ml. The results were
2.2.3. Subacute toxicity. Twenty mice (Guangzhou, Guangdong, China) were randomized into an experiment group and a control group, 50% male in each group. The extract solution was injected in the experiment group at an intraperitoneal 3
X Li et al
Biomed. Mater. 9 (2014) 045010
Figure 2. MTT assays for cell proliferation of L-929 cell cultured in different composite groups for 1, 2 and 3 days.
evaluated based on the common clinical signs and observations as specified in ISO 10993-11-2009 [23]. Animal body weights were also recorded pre-experiment, and at 1 and 2 weeks postexperiment. Furthermore, clinical pathology, organ weights, and histopathology were evaluated at 2 weeks post-experiment.
with physiological saline to remove any residual bone and bone marrow. The 80 legs of the 40 rats were randomized into 4 even groups (20 legs in each group), the defects in these 4 groups were filled with different materials: group A (nothing for empty control), group B (CaS), group C (5% Sr-CaS) and group D (10% Sr-CaS). The defects were filled flush to the anterior cortex manually with paste-form material before being allowed to set in situ. After surgery, the skin was carefully sutured and injected with antibiotics (3% Penicillin). All mice recovered well from surgery, and were housed separately in plastic cages for 12 weeks. Food and water were supplied ad libitum. Digital radiographs were taken immediately, 4, 8 and 12 weeks post-operation under anesthesia using an Oralix AC Densomat x-ray machine (Gendex Dental System, Milan, Italy). The tibias were harvested at 4, 8 and 12 weeks postoperatively for histological and micro-computed tomography evaluations.
2.2.4. Hemolysis test. The original extracts and diluted anticoagulated fresh rabbit blood were used in the test. According to the hemolysis test protocol, the test was performed in three groups: rabbit blood plus the extract in experimental group, rabbit blood plus distilled water as a positive control and rabbit blood plus physiological saline as a negative control. After 0.2 ml rabbit blood was gradually added into the extract, the centrifuge tube was placed for water bath for 1 h at 37 °C. Subsequently, 5 min centrifugation was performed at 3000 revolutions s−1. After that, the OD value at 540 nm of the supernatant was determined by a spectrometer. The rate of hemolysis was finally calculated as described in ISO 109934-2009 [24].
2.3.2. Micro-computed tomography. 20 rats (40 legs) euthanized at 12 weeks postoperatively were used for micro-computed tomography. The morphology of the reconstructed tibial cortex was assessed using an animal micro-CT scanner (eXplore Locus, GE Healthcare Biosciences, London, UK). Briefly, the specimens were scanned with a 55 kVp energy setting, intensity of 145 mA with 200 ms acquisition time and no frame average in high-resolution mode which provided a voxel resolution of 12 µm. After micro-CT scan, the defect region was identified by a contour as a traced region of interest (ROI), the relative measurements of which were calculated, including bone mineral densities (BMDs) and bone volume/ total volume (BV/TV).
2.3. Implantation in rats 2.3.1. Bone defect animal experiments. Forty-eight week-
old male Wistar rats weighing 200–250 g were used to create bone defect models as described previously [25, 26]. The general anesthesia was induced by intraperitoneal injection of Nembutal at 40 mg kg−1 body weight. The skin over the proximal tibia was incised and the periosteum was cleared using a periosteal elevator. A defect 3 mm wide and 5 mm long was created using a micro-burr with a 2.5 mm tip, starting 10 mm below the articular surface in the anteromedial cortex at both tibias. The defects and intramedullary canals were flushed 4
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Figure 3. The changes regarding body weight (a), hematological indexes ((b)–(e)) or organ weight ((f) and (g)) between the experiment and control groups. HCT, hematokrit (L L−1); MCV, mean corpuscular volume (fl); PLT, platelet count (×1010 L−1); HGB, hemoglobin (×10 g L−1); RBC, red blood cell (×1012 L−1); WBC, white blood cell, (×109 L−1); BUN, blood urea nitrogen (mmol L−1); CHO, total cholesterol (mmol L−1); GLU, glucose (mmol L−1); TG, triglyceride (mmol L−1); ALT, alanine aminotransferase (U L−1); AST, aspartate amino transferase (×10 U L−1); CRE, creatinine (µmol L−1); TP, total protein (g L−1); body weight and organ weight were all measured in grams.
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Figure 4. Fourteen days after the first injection: no inflammation or necrotic tissue was observed in the histological analysis of the liver ((a) and (b)), heart ((c) and (d)), spleen ((e) and (f)), lung ((g) and (h)) and kidney ((i) and (j)) The left column was the control group and the right was the experiment group (hematoxylin and eosin [H&E] stain, × 100 magnification). 6
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Table 2. Results of hemolysis test.
Group
OD (Mean ± SD)
Sr-CaS Negative group Positive group
0.050 ± 0.002 0.023 ± 0.001 0.656 ± 0.012
3.2. Biocompatibility test Haemolysis rate (%)
3.2.1. Cytotoxicity. The cell viability of the extracted solution
of the Sr-CaS composite measured by MTT assay at 1, 2 and 3 days after cell culture is shown in figure 2. There were no significant differences regarding the growth rate among the experimental and negative control groups (P > 0.05). Not surprisingly, the positive control group presented the least cell viability which was significantly lower than that in all the other groups (P
