Colloids and Surfaces B: Biointerfaces 130 (2015) 149–156

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Three-dimensional zinc incorporated borosilicate bioactive glass scaffolds for rodent critical-sized calvarial defects repair and regeneration Hui Wang a,1 , Shichang Zhao b,1 , Wei Xiao c , Xu Cui a , Wenhai Huang a , Mohamed N. Rahaman c , Changqing Zhang b,∗∗ , Deping Wang a,∗ a

School of Materials Science and Engineering, Tongji University, Shanghai 2001804, China Department of Orthopedic Surgery, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China c Department of Materials Science and Engineering, Missouri University of Science and Technology, Rolla, MO 65409, USA b

a r t i c l e

i n f o

Article history: Received 16 January 2015 Received in revised form 15 March 2015 Accepted 25 March 2015 Available online 3 April 2015 Keywords: Borosilicate bioactive glass Scaffolds Zinc-doped Bioactivity Osteogenesis

a b s t r a c t The biomaterials with high osteogenic ability are being intensively investigated. In this study, we evaluated the bioactivity and osteogenesis of BG-Zn scaffolds in vitro and in vivo with a rodent calvarial defects model. Zinc containing borosilicate bioactive glass was prepared by doping glass with 1.5, 5 and 10 wt.% ZnO (denoted as BG-1.5Zn, BG-5Zn and BG-10Zn, respectively). When immersed in simulated body fluid, dopant ZnO retarded the degradation process, but did not affect the formation of hydroxyapatite (HA) after long-period soaking. BG-Zn scaffolds showed controlled release of Zn ions into the medium for over 8 weeks. Human bone marrow derived stem cells (hBMSCs) attached well on the BG-1.5Zn and BG-5Zn scaffolds, which exhibited no cytotoxicity to hBMSCs. In addition, the alkaline phosphatase activity of the hBMSCs increased with increasing dopant amount in the glass, while the BG-10Zn group showed over-dose of Zn. Furthermore, when implanted in rat calvarial defects for 8 weeks, the BG-5Zn scaffolds showed a significantly better capacity to regenerate bone tissue compared to the non-doping scaffolds. Generally, these results showed the BG-Zn scaffolds with high osteogenic capacity will be promising candidates using in bone tissue repair and regeneration. © 2015 Published by Elsevier B.V.

1. Introduction In spite of rapid development in biomaterials and biotechnology, there is still a major challenge for the treatment of critical-sized bone defects [1]. To address this issue, new bioactive materials have been fabricated via incorporation of biological essential elements, such as calcium, phosphorus, copper and zinc [2–5], which can enhance bone formation and mineralization meanwhile promote the osseointegration process [4,6]. Zinc is known to play an important role in bone metabolism [7]. Previous studies have indicated that Zn possesses stimulatory effects on bone formation, ability to promote the expression and maintenance of osteoblastic phenotypes in vitro [8–10]. The effects

∗ Corresponding author. Tel.: +86 21 69582134. ∗∗ Corresponding author. Tel.: +86 21 64369181. E-mail addresses: [email protected] (C. Zhang), [email protected] (D. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2015.03.053 0927-7765/© 2015 Published by Elsevier B.V.

are particularly obvious for Zn containing materials including bone cements [11], coatings [12] and bioactive glasses [13,14]. On the other hand, Zn is a highly selective inhibitor of osteoclastic bone resorption in vitro [15]. Incorporation of Zn into bioactive materials has shown an enhancement of the proliferation and osteogenic differentiation of osteoblast, endothelial and neuronal cells [4,16]. Furthermore, there are some cellular and molecular evidences that incorporation of Zn into biomaterials could up-regulate the expression of osteoblastic relative genes such as alkaline phosphatase (ALP), collagen type I (Col-I), osteocalcin (OCN), and osteopontin (OPN). Up-regulation of these genes could further promote extracellular matrix mineralization by increasing collagen secretion synthesis and calcium deposition [17,18]. Bioactive materials such as 45S5 bioglass and some other composition based on silicate or phosphate systems have been widely used in bone tissue engineering [19–21]. The excellent bone-bonding ability arises from the high rate of formation of hydroxyapatite (HA) at the surface of the material after reaction with the surrounding biological fluids [20]. In addition, previous studies have shown that the ionic products of bioactive glass can

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stimulate gene expression and proliferation of human osteoblasts [22,23]. Recently, it has been reported that borosilicate bioactive glasses had controllable HA conversion rate and offered an attractive delivery system to release inorganic ions for healing bone defects [24,25]. Studies have also shown that the borate glass could stimulate rapid bone formation in rat tibial defects and rat calvarial defects [26,27]. Despite the established roles of Zn in bone metabolism, the feasibility of Zn containing biomaterials in clinical applications still relies on many factors, especially safety issues associated with the zinc content. On the other hand, the effect of Zn on the degradation and bioactivity of borosilicate bioactive glass in simulated body fluid (SBF) and the response of human bone marrow derived stem cells (hBMSCs) has not been studied, and no study on the properties of Zn-doped borosilicate bioactive glass in vitro or in vivo has yet been reported. In this study, it was hypothesized that Zn-doped borosilicate bioactive glass scaffolds could stimulate osteogenesis, which would be of great interest for applications in bone tissue engineering. The objective of the present study was to create porous 3-dimensional (3D) scaffolds with borosilicate bioactive glasses doped with varying amounts of Zn (1.5, 5 and 10 wt.% ZnO), and to evaluate the effects of dopant Zn on degradation and bioactivity of borosilicate bioactive glass. In vitro response of hBMSCs to the BG-Zn scaffolds was also evaluated. Furthermore, the influence of introduction of Zn into glass on the ontogenesis in osseous defects was systematically investigated using rodent calvarial defects model in vivo. 2. Materials and experiments 2.1. Preparation of BG-Zn scaffolds The bioactive glass scaffolds were created using a foam replication method as described in detail previously [28]. The parent bioactive glass (designated BG) had a borosilicate composition (6Na2 O, 8K2 O, 8MgO, 22CaO, 36B2 O3 , 18SiO2 , 2P2 O5 ; mol%). The glasses composed of BG and BG doped with 1.5, 5 and 10 wt.% ZnO (denoted as BG-1.5Zn; BG-5Zn, and BG-10Zn, respectively) were prepared using conventional melting and casting techniques. Briefly, a mixture of the requisite amounts of analytical grade ZnCO3 ·2Zn(OH)2 ·H2 O, Na2 CO3 , K2 CO3 , CaCO3 , H3 BO3 , SiO2 , (MgCO3 )4 ·Mg(OH)·25H2 O and NaH2 PO4 ·2H2 O (Sinopharm Chemical Reagent Co., Ltd., China) was heated for 1 h at 1200 ◦ C to form a molten glass. The glass frit was ground and sieved to obtain particles of average size ∼50 ␮m. Then a slurry was prepared by mixing 52.6 wt.% bioactive glass particles, 3.5 wt.% ethyl cellulose (analytical grade, Sinopharm Chemical Reagent Co., Ltd., China) and 43.9 wt.% anhydrous ethanol. A polyurethane foam (50 pores per inch) was coated by immersing in the slurry. After removal from the slurry, the coated foam was dried for 8 h in air at room temperature (RT) and heated for 2 h at 450 ◦ C (heating rate = 1.5 ◦ C/min) and then for 2 h at 550 ◦ C (heating rate = 2.5 ◦ C/min) to sinter the glass particles into a dense 3D network. 2.2. Characterization of the degradation and bioactivity of BG-Zn scaffolds The porosity of the as-prepared scaffolds was measured according to Archimedes’ principle. Degradation and conversion of the as-fabricated scaffolds were evaluated as a function of immersion time in SBF, which was prepared according to Kokubo’s method [29]. The weight loss of the scaffolds was measured as a function of time and used as a measure of HA conversion of the glass as described previously [30]. A ratio of 1 g of scaffold to 100 ml of SBF was used in all of the conversion experiments. The concentration of

released ions in SBF solution was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Optima 2100 DV; USA). The fresh immersion solution will be changed at each time point after ICP test. Three samples for each group were used in the immersion test. After the immersion, these samples were mixed together by 1:1:1 (v/v/v), then tested by ICP. The reacted scaffolds were washed with deionized water, then with ethanol, dried, coated with gold, and examined with a fieldemission scanning electron microscope (FESEM, Quanta 200 FEG). Some scaffolds were also embedded in poly(methyl methacrylate) (PMMA), ground to form a flat surface, coated with gold, and examined with a FESEM (Hitachi S-4700; Tokyo, Japan) equipped with an energy-dispersive X-ray (EDX) spectrometer (Apollo X; EDAX, Inc.). EDX analysis was used to examine compositional changes in the scaffolds due to the bioactive glass conversion. The immersed scaffolds were crushed into glass powder for the XRD analysis. XRD was performed at a scanning rate of 1◦ min−1 in the range of 10–80◦ . 2.3. In vitro cellular evaluations of BG-Zn scaffolds 2.3.1. Attachment and morphology of hBMSCs on BG and BG-Zn scaffolds The human bone marrow derived stem cells (hBMSCs) used were supplied by the Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine. The scaffolds were sterilized by heating for 2 h at 180 ◦ C in a dry atmosphere. After sterilization, the four scaffold groups were seeded with 105 hBMSCs and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO; Invitrogen Pty Ltd., Australia) supplemented with 10% fetal calf serum (FCS) (In Vitro Technologies, Australia) in 24-well culture plates at 37 ◦ C in a humidified atmosphere of 5% CO2 . After 2-day culturing, scaffolds with hBMSCs were washed with phosphate buffered saline (PBS) and placed in 2.5% glutaraldehyde in PBS at 4 ◦ C for 1 h. Then, these fixed samples were rinsed with PBS, post-fixed in 1% osmium tetroxide in PBS, dehydrated with a graded series of ethanol, then air dried. The morphology of the attached hBMSCs on BG and BG-Zn scaffolds was examined using FESEM. 2.3.2. Proliferation and ALP activity of hBMSCs cultured on BG and BG-Zn scaffolds A cell viability assay was used to evaluate the proliferation of the hBMSCs incubated on the fabricated scaffolds (Cell Counting Kit-8 (CCK-8); Dojindo Molecular Technologies, Inc., Japan). 104 hBMSCs were cultured (n = 6) in BG and BG-Zn scaffolds using the procedure described above for 1, 3 and 7 days. Subsequently, 360 ␮l of culture medium and 40 ␮l of CCK-8 solution (9:1) were added to each well at each time point and the system was incubated at 37 ◦ C for 4 h. Aliquots (100 ␮l) were taken from each well and transferred to a fresh 96-well plate. The absorbance of the samples was measured at 450 nm with a spectrophotometric microplate reader (Bio-Rad 680, USA). The results were expressed as the optical density of the aliquots minus the absorbance of the blank wells. The differentiation of hBMSCs toward the osteogenic lineage was ultimately demonstrated seeding 105 cells (n = 6) on each scaffold. The alkaline phosphatase (ALP) activity was measured at 7 and 14 days. After culturing, the medium was decanted, and the scaffolds were rinsed with PBS and 50 mM Tris buffer, then lysed in 200 ␮l 0.2% Triton X-100. Lysates were sonicated after being centrifuged at 15,000 rpm for 20 min at 4 ◦ C. Finally, 50 ␮l supernatant was mixed with 150 ␮l working solution according to the manufacturer’s protocol (Beyotime, China). The results were measured at 405 nm by a plate reader (Bio-Rad 680, USA). The ALP activity was calculated from a standard curve after normalizing to the total protein content and the results were expressed in millimoles of p-nitrophenol produced per minute per milligram of protein.

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Fig. 1. FESEM image of (a) as fabricated and (b) after 90 days immersed BG-5Zn scaffold; (c) EDX element mapping for the distribution of Ca, P and Zn in the cross section of a BG-5Zn scaffold before and after immersion in SBF for 90 days.

2.4. In vivo evaluate of new bone formation in rat critical-sized calvarial defects 2.4.1. Animals and surgery All animal surgical procedures were approved by the Animal Research Committee of the Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine. 12 male Sprague-Dawley rats (12 weeks old; body weight 250–300 g) were used for the present experiments. Based on cell culture results, the BG-5Zn scaffolds were selected for evaluation and the BG scaffolds served as control. The rats were anaesthetized with pentobarbital (Nembutal 3.5 mg/100 g). With sterile instruments and aseptic techniques, a 1.0–1.5 cm sagittal incision was made on the scalp. A critical-size defect (5 mm in diameter) was created in the central area of each parietal bone by a 5 mm electric trephine (Nouvag AG, Goldach, Switzerland) under constant irrigation with sterile 0.9% saline. Then, BG and BG-5Zn scaffolds (n = 12) were implanted in these defects. After that, the soft tissues were repositioned and sutured. Each animal was given an intramuscular injection of antibiotics post-surgery and allowed free access to food and water. They were monitored daily for potential complications. 2.4.2. Micro-computed tomography (micro-CT) Micro-CT was carried out to quantify new bone formation in calvaria defects. The implanted scaffolds were harvested after 8 weeks post-surgery, fixed with 4% paraformaldehyde, and

scanned by micro-CT (Skyscan 1176, Kontich, Belgium) at a resolution of 18 ␮m. 3D images were then created, and the percentage of the bone mineral density (BMD) and new bone volume to total bone volume (BV/TV) were analyzed using software.

2.4.3. Sequential fluorescent labeling The rate of new bone formation and mineralization were also evaluated by a polychrome sequential fluorescent labeling method. At 2, 4 and 6 weeks after surgery, the animals were given an intraperitoneal injection of fluorochromes under ether anesthesia as following: 25 mg/kg tetracycline (TE, Sigma, USA), 30 mg/kg alizarin reds (AL, Sigma, USA), and 20 mg/kg calcein (CA, Sigma, USA).

2.4.4. Histology The harvested samples were dehydrated in a graded series of ethanol (75–100%). The undecalcified specimens were embedded in methyl methacrylate and the sagittal sections of the central segment were cut, ground, and polished to a thickness of ∼40 mm. The sections were stained with Van Gieson’s picrofuchsin to identify new bone formation. The glass scaffold stained in black and new bone stained in red. The amount of bioactive glass scaffold and new bone were calculated as a percentage of the total defect area from images of the stained sections by Image Pro PlusTM (Media Cybernetics, Silver Springs, MD).

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Fig. 2. (a) XRD patterns of as fabricated scaffolds; (b) XRD patterns of BG-5Zn scaffolds as a function of immersion time in SBF. The XRD pattern of a reference hydroxyapatite (JCPDS 72-1243) is shown for comparison.

2.5. Statistical analysis Statistics were performed using ANOVA for independent samples. All data were expressed as mean ± standard deviation. Differences were considered significant for p < 0.05. 3. Results 3.1. Characterization and the degradation of as-fabricated BG-Zn scaffolds The fabricated BG and BG-Zn scaffolds had a human trabecularlike microstructure, a porosity of 89 ± 5%, 83 ± 4%, 92 ± 7%, 80 ± 5% for BG, BG-1.5Zn, BG-5Zn and BG-10Zn scaffolds respectively, with pore sizes of 200–400 ␮m. There were no marked differences in the microstructure of the four scaffold groups and, consequently, only a FESEM image of BG-5Zn scaffold was shown for brevity (Fig. 1a). After 90 days of immersion, the surface of BG-5Zn became rougher, with grain-like assembled particles (Fig. 1b). Fig. 1c showed the typical microstructure of the initial and post-immersion BG-5Zn scaffold strut. After 90 days of immersion in SBF, the surface of BG-5Zn was homogenously covered with

calcium phosphate (CaP) layer. A closer look at the inner part of the scaffold (inner part of the struts) in a scaffold cross-section showed dissolution ions released to immersion solution. CaP enrichment on the scaffold surface further indicated smaller round-shaped precipitates marked as HA. XRD analysis in Fig. 2a showed BG and BG-Zn scaffold glass powders had broad reflection at 30◦ , typical of amorphous glass. In comparison, XRD patterns of the BG-5Zn scaffold immersed for 30 and 90 days in SBF showed peaks that corresponded to a reference HA (JCPDS 72-1243), confirming the conversion of the scaffolds to HA (Fig. 2b). The same trend was observed for the other three groups of scaffolds (results not shown). In general, the weight loss, pH and release of Zn and B ions showed similar trends. A more rapid increase in the first 10–14 days was followed by a much slower increase thereafter. When the experiments were terminated at 56 day, the final weight loss was 49.0 ± 2.4%, 46.8 ± 3.1%, 44.7 ± 2.9% and 40.4 ± 1.7%, respectively, for the BG, BG-1.5Zn, BG-5Zn and BG10Zn scaffolds (Fig. 3a). The theoretical weight loss of borosilicate scaffolds is 47.8%, assuming that all the boron was released and all the calcium in the BG glass was converted to HA. These results suggested that BG and BG-Zn scaffolds were almost completely

Fig. 3. (a) Weight loss for the four groups of BG and BG-Zn scaffolds; (b) pH changes of the immersion medium; mean ± SD; n = 3. (c and d) Concentration of Zn and B ions released from the glass scaffolds into the medium as a function of immersion time of the scaffolds in SBF.

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Fig. 4. FESEM images of hBMSCs cultured on the four groups of scaffolds for 48 h: (a) BG; (b) BG-1.5Zn; (c) BG-5Zn; (d) BG-10Zn.

converted to HA by day 56. When compared with the BG scaffolds, the BG-Zn scaffolds showed a significant decrease (Fig. 3b) of the pH changes of the soaking SBF. At day 56, the pH was 9.02 ± 0.07, 8.73 ± 0.10, 8.53 ± 0.07 and 8.40 ± 0.12, respectively, for the BG, BG-1.5Zn, BG-5Zn and BG-10Zn scaffolds. The highest released amount of Zn ions was at day 1, and the release of Zn had mostly ceased by day 14, with the exception of BG-10Zn scaffolds. The highest release amount was 7.8, 13.8, and 29.4 ppm for BG-1.5Zn, BG-5Zn and BG-10Zn scaffolds (Fig. 3c), respectively. At day 56, the cumulative amount of Zn released from the scaffolds was 45.90, 120.8, and 213.8 ppm (cumulated from the interval concentration in Fig. 3) taking the percentage of total amount of doping Zn 38.1%, 30.1% and 26.6%. The total amount of B released into the medium was 692, 635, 579, and 511 ppm (cumulated from the interval concentration in Fig. 3) taking the percentage of total amount of B in the as-fabricated BG, BG-1.5Zn, BG-5Zn, and BG-10Zn scaffolds 97.4%, 90.7%, 85.8% and 79.6% respectively. Results showed that introduction of Zn into borosilicate glass retarded its degradation process, but did not affect the formation of HA after long-period of soaking from results of XRD. 3.2. Adhesion and proliferation of hBMSCs on BG and BG-Zn scaffolds FESEM images of hBMSCs cultured on the surfaces of the four group scaffolds for 48 h showed that the cells attached and spread well (Fig. 4). The morphology of the hBMSCs also showed clear and prominent filopodia.

As determined by CCK-8 assay (Fig. 5a), the proliferation of hBMSCs increased remarkably in a time dependent manner, except the BG-10Zn group. Significant increase of cell proliferation of hBMSCs was found in BG-5Zn group compared to that in the BG group on day 3, 7 (p < 0.05). However, cell proliferation in BG-10Zn group decreased significantly on day 7 (p < 0.05). The relative ALP activity of hBMSCs for BG-5Zn group showed a substantial increase at both incubation times (7 and 14 days) when compared to the cells cultured on the undoped BG scaffolds (Fig. 5b). A significant decrease of ALP activity in hBMSCs cultured in the BG-10Zn group for 14 days was observed (p < 0.05). No significant difference of ALP activity between day 7 and day 14 was noted for BG-1.5Zn group (p > 0.05). These results showed that at the concentrations (1.5–5 wt.% ZnO) used, the Zn in the glass was not toxic to the hBMSCs and the BG-5Zn scaffolds had the best capacity to support the osteogenic differentiation of the hBMSCs. 3.3. Evaluation of osteogenesis in vivo Reconstructed 3D and 2D micro-CT images of rat calvarial defects implanted for 8 weeks with the BG and BG-5Zn scaffolds were shown in Fig. 6. The images showed a higher amount of new bone in the defects implanted with the BG-5Zn scaffolds than that of BG group. The defects implanted with the BG scaffolds showed macropores in the scaffolds and clear gaps in the edge area between scaffolds and host bone than BG-5Zn group. Results of quantitative analysis of the micro-CT images indicated that the bone mineral density (BMD) in the BG-5Zn scaffolds (522 ± 67 mg/cm3 ) was significantly higher than in the BG scaffolds (261 ± 54 mg/cm3 )

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Fig. 5. Quantitative measurement of cell proliferation by (a) CCK-8 assays and (b) ALP activity of hBMSCs cultured for 7 and 14 days on the four groups of bioactive glass scaffolds shown. Mean ± SD; n = 6. *Significant difference when compared to BG (p < 0.05).

(Fig. 6b). The bone volume divided by the total volume (BV/TV) determined by the micro-CT images showed a similar trend as that of BMD (Fig. 6c). The BV/TV for the defects implanted with the BG5Zn scaffolds (29.1 ± 3%) was significantly higher than that of the BG scaffolds (16.4 ± 5%). Together, these results suggested that the BG-5Zn scaffolds had a higher ability to integrate with the host bone than the BG scaffolds. Fig. 7 showed data for the new bone formation and mineralization in the defects implanted with the BG and BG-5Zn scaffolds, which were determined by fluorochrome-labeling analysis. After 2 weeks, tetracycline (yellow) was found to be deposited onto a broader area adjacent to the implants in the BG group, and even more adjacent to BG-5Zn, which exhibited the most intense and widely distributed yellow fluorescence. Furthermore, alizarin red (4 weeks) was found to extend along the BG-5Zn surface, indicating a larger bone formation rate than the BG group. At a later time point (6 weeks), calcein (green) was incorporated into the bone closest to the implant coatings, demonstrating similar patterns. According to the combined images collected at different time points, the newly formed bone spread throughout the entire region, from the implant surface toward existing bones, and the most distinctive fluorescent lines were produced by BG-5Zn scaffolds. A higher amount of new bone in the defects implanted with the BG-5Zn scaffolds than in the defects implanted with the BG scaffolds at 8 weeks (Fig. 8a), was shown by the images of

non-decalcified sections stained with Van Gieson’s picrofuchsin. Quantitative analysis of these images indicated that the percent of new bone area in the defects implanted with the BG-5Zn (31 ± 3%) was obviously higher than that of the BG scaffolds (20 ± 4%) (Fig. 8b). The histomorphometric results showed that the BG5Zn scaffolds had significantly better capacity to heal rat calvarial defects when compared to BG scaffolds. 4. Discussion As a local regulator of bone cells, zinc has stimulatory effects on bone metabolism in vitro and in vivo [31,32]. Therefore, it is crucial to explore the use of zinc in biomaterials as a therapeutic agent to induce rapid bone healing and bone growth, particularly pertaining to dentistry and orthopedics [33]. In this study, we successfully fabricated Zn incorporated borosilicate bioactive glass scaffolds by a polymer foam replication technique. The as-fabricated BG-Zn and BG scaffolds have similar microstructure to human trabecular, which is known to be favorable for supply of oxygen and nutrients, attachment, migration, and proliferation of bone-forming cells, vascularization and bone ingrowth [34]. The incorporation of 1.5–10 wt.% ZnO into a parent borosilicate bioactive glass resulted in small reductions in the degradation of porous 3D scaffolds as measured by the weight loss of the scaffolds, the pH of the medium, and the concentration of boron and Zn ions released into the medium (Fig. 3).

Fig. 6. Micro-CT evaluation of bone regeneration in the rat calvarial defects implanted with the BG and BG-5Zn scaffolds at 8 weeks post-implantation. (a) Top and crosssectional views of reconstructed images; (b and c) bone mineral density (BMD) and bone volume/total volume (BV/TV) in the defects implanted with the scaffolds and in the unfilled defects. Mean ± SD; n = 3. *Significant difference between groups (p < 0.05).

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Fig. 7. New bone formation and mineralization, determined by fluorochrome-labeling analysis, in rat calvarial defects implanted with the BG and the BG-5Zn scaffolds. Column 1 (yellow) showed tetracycline at week 2; column 2 (red) showed alizarin red at week 4; column 3 (green) showed calcein at week 6; column 4 represents merged images of the three fluorochromes for the same group, and colum 5 represents merged images from a plain microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

ZnO can act as both network-modifying oxide and intermediate oxide in glass structure [35]. Part of Zn2+ can facilitate the charge balanced isomorphic replacement of SiO4 or BO4 tetrahedra in the glass structure, with ZnO4 tetrahedra; the remaining Zn2+ will act as a network modifier serving to increase the disruption of the glass [36]. While the high agglutination effect of zinc ions could cause the formation of atomic cluster which could be the glass structure in metallic glass [37], when some Zn ions disrupt the network of B O or Si O. Therefore, the incorporation Zn into glass will stabilize the glass system, leading to a slower degradation process. Similar results show that Zn promoted the crystallization of SiO2 and wollastonite in these glass ceramic samples, which retarded the biological mineralization process in glass-ceramics prepared by calcining sol–gel derived bioactive glasses [38]. On the other hand, the easy doping with Zn in borosilicate glass during manufacture resulted in advantageous sustained release of Zn ions from the BG-Zn scaffolds into the liquid medium for more than 8 weeks. At day 56, the cumulative amount of Zn released from the scaffolds was 45.90, 120.8, and 213.8 ppm taking the percentage of total amount of doping Zn 38.1%, 30.1% and 26.6% and the highest release amount was 7.8, 13.8, and 29.4 ppm for BG1.5Zn, BG-5Zn and BG-10Zn scaffolds respectively (Fig. 3c). The

data indicated that the release of Zn from the borosilicate bioactive glass scaffolds used in this study can be controlled by modifying the concentration of Zn in the as-prepared glass. However, high concentrations of Zn induce cell death, possibly through the production of free radicals: antioxidants blunt such zinc toxicity [43]. At the concentrations of 1.5–5 wt.%, the incorporation of Zn into the borosilicate bioactive glass was not toxic to hBMSCs when cultured on the glass scaffolds, while the proliferation and the cell viability have been inhibited with time as shown in Fig. 5 for the BG-10Zn group. Results also showed that BG-5Zn scaffolds exhibited a significant increased ALP activity compared to the BG scaffolds (Fig. 5b), indicating that the BG-5Zn scaffold was capable of supporting the differentiation of hBMSCs. The results presented here were consistent with the previous study in zinc-containing medium [39], which indicated a zinc concentration between 10 ␮M and 250 ␮M (i.e. 0.65 ppm and 16.25 ppm) as the main cause of osteoclastogenesis suppression. Holloway et al. [40] noticed that treatment with zinc concentration lower than 10−4 mol/L (6.5 ppm) had no effect on osteoclast activity. In order to explore the potential clinical applications, an in vivo study on the ability of the scaffolds to repair a critical-sized osseous defect was performed. The results of the present study showed

Fig. 8. (a) Transmitted light images of Van Gieson picrofuchsin-stained sections of rat calvarial defects implanted with BG and BG-5Zn scaffolds at 8 weeks post-implantation. New bone appears red whereas the scaffold appears black. (b) Percent new bone area in the defects implanted with the scaffolds and in the unfilled defects. Mean ± SD; n = 3. *Significant difference between groups (p < 0.05).

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that the incorporation of 5 wt.% ZnO in the borosilicate bioactive glass scaffolds significantly enhanced bone regeneration in rat calvarial defects at 8 weeks when compared to the BG scaffolds (Figs. 6–8). Zinc is an essential trace element that is involved in numerous cellular enzymatic reactions and gene regulation through the modulation of several transcription factors [41,42]. In adults, zinc deficiency has been associated with retarded skeletal development and development of osteoporosis [43]. In MC3T3-E1 cells, zinc and zinc-chelating compounds can stimulate proliferation, ALP activity, and increase expression of several osteoblast markers, such as osteopontin (OPN) and osteocalcin (OCN) [44]. In both MSCs and osteoblastics, a ubiquitous zinc transporter, ZIP1 has been defined as a functional role for a zinc transport mechanism in osteogenic differentiation. The amount of ZIP1 will increase with the zinc amount [45]. It has been reported that the expression of OPN and periostin were up-regulated, ranging from an up regulation of ∼5- to 24-fold following overexpression of ZIP1 in MSCs [46]. The overexpression of ZIP1 in MSCs resulting in an increased gene expression of OPN may be indirectly mediated through the direct effects of zinc on the activity of vitamin-D-dependent promoters in osteoblasts [47,48]. Periostin is a cell adhesion molecule for preosteoblasts and is regulated during osteoblast differentiation. A recent study also showed that overexpression of ZIP1 in MSCs could induce up-regulated expression of Runx2 which further promoted osteogenic differentiation [49]. As a result, the highly co-expressed osteogenic factors can act synergistically to recruit hBMSCs into the bone defects, which increases cell survival and promotes cell ossification. Consequently, the BG-5Zn scaffolds show improved osteogenesis than the BG group. Although Zn has shown to be very useful in biomedical and tissue engineering in recent years, the mechanisms of Zn ions in scaffolds stimulating hBMSCs are not clear at the present time. Further studies are needed to understand those mechanisms. 5. Conclusion Here, a systematic investigation was performed on BG and BGZn scaffolds with different contents of ZnO (1.5, 5 and 10 wt.%) in vitro and in vivo. In this study, BG-Zn scaffolds were successfully synthesized by the foam replica method. When immersed in SBF, the Zn in glass retarded the degradation process of borosilicate glass. BG-Zn scaffolds have no significant cytotoxicity, support hBMSCs adhesion and proliferation, except for in high concentrations (BG-10Zn group). Importantly, when implanted in rat calvarial defects in vivo for 8 weeks, the BG-5Zn scaffolds significantly enhanced bone regeneration in the defects. These results show that the incorporation of Zn into BG scaffold has scientific merit, which is a promising strategy for repairing bone tissue defects. Acknowledgments The authors gratefully acknowledge the support by the National Natural Science Foundation, China (Grant nos. 51072133, 51272274 and 51372170) and Science and Technology Commission of Shanghai Municipality, China (Grant no. 12JC1408500). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.03. 053

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Three-dimensional zinc incorporated borosilicate bioactive glass scaffolds for rodent critical-sized calvarial defects repair and regeneration.

The biomaterials with high osteogenic ability are being intensively investigated. In this study, we evaluated the bioactivity and osteogenesis of BG-Z...
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