JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH J Tissue Eng Regen Med (2015) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2062

ARTICLE

Composites of gellan gum hydrogel enzymatically mineralized with calcium–zinc phosphate for bone regeneration with antibacterial activity Timothy E. L. Douglas1*, Magdalena Pilarz2, Marco Lopez-Heredia3, Gilles Brackman4, David Schaubroeck5, Lieve Balcaen6, Vitaliy Bliznuk7, Peter Dubruel1, Christine Knabe-Ducheyne3, Frank Vanhaecke6, Tom Coenye4 and Elzbieta Pamula2 1

Polymer Chemistry and Biomaterials (PBM) Group, Department of Organic Chemistry, Ghent University, Belgium Department of Biomaterials, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, Krakow, Poland 3 Department of Experimental and Orofacial Medicine, Faculty of Dentistry, Philipps University, Marburg, Germany 4 Laboratory of Pharmaceutical Microbiology, Ghent University, Belgium 5 Centre for Microsystems Technology (CMST), IMEC, and Ghent University, Belgium 6 Department of Analytical Chemistry, Ghent University, Belgium 7 Department of Materials Science and Engineering, Zwijnaarde, Belgium 2

Abstract Gellan gum hydrogels functionalized with alkaline phosphatase were enzymatically mineralized with phosphates in mineralization medium containing calcium (Ca) and zinc (Zn) to improve their suitability as biomaterials for bone regeneration. The aims of the study were to endow mineralized hydrogels with antibacterial activity by incorporation of Zn in the inorganic phase, and to investigate the effect of Zn incorporation on the amount and type of mineral formed, the compressive modulus of the mineralized hydrogels and on their ability to support adhesion and growth of MC3T3-E1 osteoblast-like cells. Mineralization medium contained glycerophosphate (0.05 m) and three different molar Ca:Zn ratios, 0.05:0, 0.04:0.01 and 0.025:0.025 (all mol/dm3), hereafter referred to as A, B and C, respectively. FTIR, SAED and TEM analysis revealed that incubation for 14 days caused the formation of predominantly amorphous mineral phases in sample groups A, B and C. The presence of Zn in sample groups B and C was associated with a drop in the amount of mineral formed and a smaller mineral deposit morphology, as observed by SEM. ICP–OES revealed that Zn was preferentially incorporated into mineral compared to Ca. Mechanical testing revealed a decrease in compressive modulus in sample group C. Sample groups B and C, but not A, showed antibacterial activity against biofilm-forming, methicillin-resistant Staphylococcus aureus. All sample groups supported cell growth. Zn incorporation increased the viable cell number. The highest values were seen on sample group C. In conclusion, the sample group containing the most Zn, i.e. group C, appears to be the most promising. Copyright © 2015 John Wiley & Sons, Ltd. Received 15 January 2015; Revised 22 April 2015; Accepted 4 May 2015

Keywords

hydrogel; gellan gum; mineralization; zinc; composite; antibacterial

1. Introduction Gellan gum (GG) is a cheap, anionic polysaccharide consisting of a repeating unit of β-D-glucuronic acid, βD-glucose and α-L-rhamnose in the molar ratio 1:2:1 (in

*Correspondence to: Timothy E. L. Douglas, Nano- and Biophotonics Group, Department of Molecular Biotechology, Coupure Links 653, 9000 Ghent, Belgium. E-mail: Timothy. [email protected] Copyright © 2015 John Wiley & Sons, Ltd.

the deacetylated form), which is generated biotechnologically, using bacteria, and has been used in the pharmaceutical and medical industries (Giavasis et al., 2000; Osmalek et al., 2014). GG can form hydrogels as a result of crosslinking by divalent ions (Morris et al., 2012). GG has been applied as a biomaterial in tissue engineering to support the regeneration of several tissues, including cartilage, intevertebral discs (Lee et al., 2012; Pimenta et al., 2011; Silva-Correia et al., 2011) and skin (Cerqueira et al., 2014a, 2014b). GG hydrogels have been adapted for applications in bone regeneration by the addition of inorganic particles,

T. E. L. Douglas et al.

such as bioactive glasses (Gantar et al., 2014; Kocen et al., 2014; Douglas et al., 2014b) and hydroxyapatite (Canadas et al., 2012; Manda-Guiba et al., 2012). More recently, such GG hydrogels have been enzymatically mineralized with calcium phosphate (CaP) in order to provide mechanical reinforcement and promote osteoblast adhesion and proliferation (Douglas et al., 2014a). Enzymatic mineralization of hydrogels can be realized by the addition of alkaline phosphatase (ALP) during hydrogel formation, followed by incubation in a solution containing calcium ions and glycerophosphate (GP), which is a substrate for ALP (Douglas et al., 2012; Filmon et al., 2000; Spoerke et al., 2009). ALP-mediated cleavage of phosphate from GP raises the intrahydrogel phosphate concentration, causing precipitation of insoluble CaP. In vivo studies involving implantation of ALP as a biomaterial component have not revealed any severe inflammatory reactions (Beertsen and van den Bos, 1992; Bongio et al., 2013; Doi et al., 1996; Schouten et al., 2009). The mineral formed in hydrogels by enzymatic action does not necessarily need to be pure CaP. Other metal ions, e.g. Mg2+, can be incorporated into the mineral formed by simple addition of the ions to the mineralization solution. In previous work, ALP-enriched GG hydrogels were mineralized with five different mineralization solutions of varying Ca:Mg molar ratios in order to assess the effect of magnesium on the physiochemical and biological properties of the resulting composites (Douglas et al., 2014c). Increasing magnesium concentration led to increased amorphicity of the CaP formed, and the presence of magnesium in the mineral formed enhanced the proliferation of osteoblast-like cells. In the present study, zinc was incorporated into mineral formed by the same strategy. Zinc is gaining in interest as a component of biomaterials for bone regeneration, for several reasons (Habibovic et al., 2008; Mourino et al., 2012). First, as a component of apatite-based materials, zinc has supported the proliferation of mesenchymal stem cells (MSCs) (Thian et al., 2013) and the adhesion, proliferation and osteogenic differentiation of osteoblast-like cells (Webster et al., 2002; Yang et al., 2012; Miao et al., 2007; Wang et al., 2010) and pre-osteoblasts (Storrie and Stupp, 2005). Second, as a component of tricalcium phosphate (TCP), biphasic TCP–hydroxyapatite (HA) and brushite ceramics, zinc has increased new bone formation and bone–implant contact in vivo (Kawamura et al., 2000, 2003; Li et al., 2009; Pina et al., 2010); Znenriched TCP has also stimulated ectopic bone formation (Luo et al., 2014). Third, zinc has exhibited antibacterial activity as an additive to HA (Thian et al., 2013). Fourth, there are indications that zinc can stimulate angiogenesis (Imai et al., 2010). The aim of this study was the development of enzymatically mineralized hydrogel–inorganic composites with antibacterial activity and enhanced cell adhesion, thanks to the presence of zinc. Samples were incubated in solutions of GP with three different Ca:Zn concentration ratios. The influence of the Ca:Zn concentration ratio on the physiochemical and biological properties of Copyright © 2015 John Wiley & Sons, Ltd.

enzymatically mineralized GG hydrogels was assessed. The physicochemical properties investigated included the amount and nature of mineral formed and the resistance to compressive loading. These were analysed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), selected area electron diffraction (SAED), attenuated total reflectance Fourier transform infrared spectroscopy (ATR–FTIR), inductively coupled plasma optical emission spectroscopy (ICP–OES), monitoring of the dry mass percentage, i.e. the mass fraction of the hydrogel not consisting of water, and compressive testing. The biological properties included antibacterial activity, cytocompatibility and ability to support the adhesion of osteoblast-like cells. As mentioned above, Zn has been incorporated into pure inorganic biomaterials by other authors. However, to our best knowledge, the generation of Zn-enriched hydrogel–inorganic composite biomaterials is novel. Furthermore, enzymatic mineralization of biomaterials for bone contact with metal ions other than Ca2+, e.g. Zn2+, is not well explored.

2. Materials and methods 2.1. Materials All materials, including GG (Gelzan™ CM, product no. G1910, ’Low-Acyl’, molecular weight 200–300 kDa), calcium chloride dihydrate (product no. 233506), zinc chloride (product no. 229997) and β-glycerophosphate disodium salt pentahydrate (NaGP) (product no. 50020) were obtained from Sigma-Aldrich, unless stated otherwise.

2.2. Production of ALP-loaded GG hydrogels GG hydrogel discs containing ALP were produced using the method of Oliveira et al. (2010), with some modifications, as described in previous publications (Douglas et al., 2014a, 2014c). Briefly, 16 ml aqueous 0.875% w/v GG solution at 90°C was mixed with 3.6 ml aqueous 0.169% w/v CaCl2 at 90°C, resulting in a GG–CaCl2 solution. After cooling to 50°C, 0.4 ml 25 mg/ml ALP solution was added. Final concentrations of GG, CaCl2 and ALP were 0.7% w/v, 0.03% w/v and 0.5 mg/ml, respectively. Immediately after ALP addition, 20 ml GG–ALP solution was cast in glass Petri dishes of diameter 10 cm at room temperature and left for 20 min to ensure complete gelation. Hydrogel discs 7 mm in diameter and 5 mm in thickness were cut, using a hole punch.

2.3. Incubation of hydrogels in mineralization media Hydrogel discs were immersed in three different mineralization media at room temperature. These media, denoted as A, B and C, contained identical concentrations of NaGP (0.05 m) and different concentrations of CaCl2 and ZnCl2. J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Antibacterial hydrogels mineralized with Ca–Zn phosphate

The Ca:Zn concentration ratios in A, B and C were 0.05:0, 0.04:0.01 and 0.025:0.025, respectively (all values mol/dm3); a detailed overview is provided in Table 1. The mineralization medium was changed every day. After the conclusion of mineralization (after 7, 10 or 14 days), the samples were rinsed three times in Milli-Q water.

2.4. Mineralization of gels and calculation of mass change due to mineralization The dry mass percentage, i.e. the sample weight percentage not consisting of water, served as a measure of the extent of mineral formation, as in previous publications (Douglas et al., 2012). Dry mass percentage was calculated as: weight after incubation in mineralization medium and 48 h freeze-drying/weight after incubation in mineralization medium before freeze-drying × 100; for all sample groups, n = 10.

2.5. Physicochemical and morphological characterization: SEM, TEM, SAED, ATR–FTIR, ICP–OES and Zn release studies After incubation in media A, B and C for 14 days, the morphology and molecular structure of the hydrogels was investigated using SEM, TEM, SAED and ATR–FTIR, as described previously (Gassling et al., 2013). SEM was performed using a JEOL JSM-5600 device; samples were sputtered with a thin gold layer to make them conductive. TEM and SAED were carried out using a JEM-2200FS FEG (JEOL) device set to 200 kV in conventional brightfield and SAED modes. Chromatic aberration as a result of inelastic scattering of primary electrons in thick areas of the sample was diminished by the use of an in-column omega filter. ATR–FTIR was performed using a Bio-Rad FTS 575C device; this had a ‘Golden Gate’ ATR accessory, which was fitted with a diamond crystal; the settings were: range 4000–500 cm-1, 32 scans, resolution 4 cm-1. The mass and molar concentrations of Ca, Zn and P and the Ca:P, Ca:Zn and Zn:P molar ratios were determined by ICP–OES, as described in previous publications (Douglas et al., 2014c; Gassling et al., 2013b). Briefly, samples were dissolved in 14 M analytical grade HNO3 and further diluted with 0.3 M HNO3 to obtain appropriate concentrations for analysis. Measurements were performed using a Spectro Arcos Optical Emission Spectrometer (Spectro, Germany), calibrated with standard solutions of Ca, Zn and P whose concentrations were in the range 0–15 mg/l. Table 1. Mineralization media used in this study 3

Concentration (mol/dm )

Mineralization medium

CaCl2

ZnCl2

NaGP

A B C

0.05 0.04 0.025

0 0.01 0.025

0.05 0.05 0.05

Copyright © 2015 John Wiley & Sons, Ltd.

Yttrium was added to the solutions and served as an internal standard. All measurements were performed three times. Release of Zn was studied by incubation of one sample in 2 ml cell culture medium. After 24 h, Zn concentration in the cell culture medium was determined by ICP– OES, as described previously (Douglas et al., 2014c; Gassling et al., 2013a). Measurements were performed in triplicate (n = 3).

2.6. Mechanical testing Hydrogel samples were subjected to compressive testing at a rate of displacement of 4 mm/min, up to 50% of their original height, up to a maximum load of 80 N, using a Hounsfield Universal Testing Machine H10KM equipped with a 100 N load cell, which recorded force every 0.5 s using Qmat software, as described previously (Douglas et al., 2014c). Young’s modulus under compression was calculated as compressive strength/compressive strain; more specifically, force recorded divided by the cross-sectional area/distance moved during compression, divided by the initial distance. For all sample groups, n = 4.

2.7. Sterilization of materials prior to antibacterial and cell biological testing To prepare samples for cell biological characterization, GG powder was sterilized using ethylene oxide, as described previously (Douglas et al., 2014d). All other reagents necessary for GG hydrogel formation and mineralization, including CaCl2 solution, ALP solution and mineralization media A, B and C, were sterilized by filtration under sterile working conditions.

2.8. Antibacterial testing An ‘uninfected wound model’ and an ‘infected wound model’ were used. The former simulates bacterial growth on materials placed in uninfected wounds. The latter simulates bacterial biofilm formation on materials placed in infected wounds or on microbially-contaminated materials placed in uninfected wounds. Both models have been described in detail in previous work (Brackman et al., 2011; Garcia-Fernandez et al., 2013; Douglas et al., 2014b). One, three or six hydrogel discs and a silicone disc were placed in the ‘uninfected wound model’, and one hydrogel disc and a silicone disc were placed in the ‘infected wound model’, as previously described (Douglas et al., 2014b). Growth of methicillin-resistant Staphylococcus aureus (MRSA) Mu50 was expressed as the number of colonyforming units (CFU) relative to the control (silicone discs without added hydrogel discs). Since one hydrogel disc proved sufficient to reduce MRSA growth significantly in the ‘uninfected wound model’ (see results section), only one hydrogel disc was placed in the ‘infected wound model’. For all sample groups, n = 5. J Tissue Eng Regen Med (2015) DOI: 10.1002/term

T. E. L. Douglas et al.

2.9. Cell biological characterization A commercially available MC3T3-E1 subclone 4 cell line (ATCC, USA) was used to perform in vitro cell biological characterization. The cell culture medium (CCM) used consisted of Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Germany) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine (Gibco), 5 mM β-GP and 50 μg/ml penicillin–streptomycin (Gibco). Cells were cultured in T75 cell culture flasks with 13 ml CCM in an incubator; the CCM was refreshed three times weekly. Cells were grown until confluence and then detached with 0.25% trypsin/0.02% EDTA (BioChrom, Germany) solution, and passaged. Cells were seeded on mineralized GG samples at 50 000 cells/GG sample. Briefly, 50 000 cells were suspended in 100 μl CCM and left in contact, in an incubator, with the hydrogel sample for 4 h. After this time, the wells were filled with CCM; then, after 24 h of culture, cell proliferation and cytotoxicity were measured using an Alamar Blue® assay (Invitrogen, Germany) and CellTox Green® assay (Promega, Germany), respectively. Assays were performed in triplicate (n = 3), according to the manufacturers’ instructions. Cells seeded at different densities in normal plastic wells were used as standards for comparison purposes.

Figure 1. Dry mass percentage of GG hydrogels containing 2.5 mg/ml ALP incubated for 14 days in mineralization media A, B and C; error bars show SD; *significant differences; ***p < 0.001

Table 2. ICP–OES determination of mass elemental Ca, P and Zn/unit mass of lyophilized hydrogels containing 2.5 mg/ml ALP, incubated for 7, 10 or 14 days in media A, B and C (μg element/mg sample)

2.10. Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test, using standard statistical analysis software (SPSS statistics software, IBM Corp., USA) and Origin (OriginLab Corp., USA); p < 0.05 was considered significant.

3. Results and discussion 3.1. Influence of mineralization medium on dry mass percentage and elemental composition The dry mass percentages of samples are shown in Figure. 1. Samples incubated in medium A exhibited the highest values after 7, 10 and 14 days. These values were approximately 1.5 times higher than those observed for samples incubated in media B and C. An increase was observed between days 7 and 14 for samples incubated in media A and C, but not for medium B. Elemental mass compositions of samples after 7, 10 and 14 days are presented in Table 2. Amounts of atomic Ca, P and Zn present were highest after 14 days, which is consistent with the results of dry mass percentage measurements (Figure 1). However no increase was observed between days 7 and 10. Ca:Zn, Ca:P and Zn:P molar ratios are presented in Table 3. No marked change with incubation time was observed. The reason for the superior mineral formation in sample group A is not clear; the question of the affinity of Copyright © 2015 John Wiley & Sons, Ltd.

Sample group

Ca

P Zn Incubation time in medium (days) Mean SD Mean SD Mean SD

A B C A B C A B C

7 7 7 10 10 10 14 14 14

215 44 30 193 46 26 226 62 47

27 3 9 22 5 2 13 5 5

121 66 71 109 70 61 127 94 91

16 b.d.l. b.d.l. 2 134 7 18 163 41 12 b.d.l. b.d.l. 6 144 12 3 143 9 7 b.d.l. b.d.l. 6 199 10 11 217 23

Values are presented as mean ± SD (n = 3); b.d.l., below detection limit of apparatus, hence incalculable.

Table 3. ICP–OES determination of elemental molar ratios Ca:Zn, Ca:P and Zn:P in lyophilized hydrogels containing 2.5 mg/ml ALP, incubated for 7, 10 or 14 days in media A, B and C

Sample group A B C A B C A B C

Molar elemental ratios Incubation time in Ca:Zn Ca:P Zn:P medium Ratio SD Ratio SD Ratio SD (days) 7 7 7 10 10 10 14 14 14

b.d.l. 0.54 0.29 b.d.l. 0.53 0.29 b.d.l. 0.51 0.36

b.d.l. 0.003 0.02 b.d.l. 0.007 0.01 b.d.l. 0.01 0.006

1.37 0.52 0.32 1.37 0.51 0.33 1.38 0.51 0.40

0.007 b.d.l. b.d.l. 0.007 0.96 0.02 0.02 1.09 0.003 0.02 b.d.l. b.d.l. 0.009 0.97 0.005 0.02 1.12 0.02 0.009 b.d.l. b.d.l. 0.009 1.00 0.008 0.01 1.13 0.02

Values are presented as mean ± SD (n = 3); b.d.l., below detection limit of apparatus, hence incalculable. J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Antibacterial hydrogels mineralized with Ca–Zn phosphate

GG for Zn2+ and Ca2+ arises. It has been reported that GG has a higher affinity for Zn2+ than Ca2+ (Grasdalen and Smidsrod, 1987). Hence, one would expect this to favour higher mineral formation in sample groups B and C, which contain Zn. However, the amount of mineral formed in these groups was lower than that formed in the Zn-free sample group A. Differences in release of ALP from GG hydrogels may also exert an influence. A previous study has shown that ALP is released from GG hydrogels (Douglas et al., 2014a). No ionic interaction between GG and ALP was expected, due to the anionic

nature of GG and the low isoelectric point of ALP ( 400 μM have been reported to be toxic to MC3T3-E1 cells, as reported by Brauer et al. (2011). The concentrations of Zn

released from sample groups B and C after 24 h (Table 4) were above the concentrations used by Seo et al. (2010) but lower than the toxic concentrations reported by Brauer et al. (2011). Hence, it is conceivable that the release of Zn from sample groups B and C resulted in higher signals (Figure 7).

4. Conclusion GG hydrogels were enzymatically mineralized with an inorganic phase consisting of calcium, zinc and phosphates. Zn was incorporated into the mineral formed to a greater extent than Ca. The inorganic phase was predominantly amorphous, regardless of the amount of Zn present. The presence of Zn caused a reduction in size of the mineral deposits formed. The sample groups containing Zn, i.e. groups B and C, showed antibacterial activity. An increase in numbers of MC3T3-E1 osteoblast-like cells after 1 day was observed in sample group C. Of the three sample groups tested, group C seems to be the most promising, due to superior antibacterial activity and cell numbers. In summary, incorporation of Zn into enzymatically mineralized hydrogels can endow antibacterial activity and also promote the adhesion of bone-forming cells.

Conflict of Interest The authors declare no conflicts of interest.

Acknowledgement T.E.L.D. acknowledges the Research Foundation Flanders (FWO) for support in the framework of a postdoctoral fellowship.

References Beertsen W, Van den Bos T. 1992; Alkaline phosphatase induces the mineralization of sheets of collagen implanted subcutaneously in the rat. J Clin Invest 89: 1974–1980. Copyright © 2015 John Wiley & Sons, Ltd.

Brackman G, Cos P, Maes L et al. 2011; Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo. Antimicrob Agents Chemother 55: 2655–2661.

Brauer DS, Gentleman E, Farrar DF et al. 2011; Benefits and drawbacks of zinc in glass ionomer bone cements. Biomed Mater 6: 045007.

J Tissue Eng Regen Med (2015) DOI: 10.1002/term

Antibacterial hydrogels mineralized with Ca–Zn phosphate Brun LR, Traverso A, Rigalli A 2009; Aggregation and inhibition of rat intestinal alkaline phosphatase by high concentrations of calcium. Reversibility of the processes. J Enzyme Inhib Med Chem 24: 691–696. Canadas R, Pereira DR, Silva-Correia J et al. 2012; Novel bilayered gellan gum/gellan gum–hydroxyapatite scaffolds for osteochondral tissue engineering applications. J Tissue Eng Regen Med 6: 16. Cerqueira MT, Da Silva LP, Santos TC et al. 2014a; Human skin cell fractions fail to selforganize within a gellan gum/hyaluronic acid matrix but positively influence early wound healing. Tissue Eng A 20: 1369–1378. Cerqueira MT, Da Silva LP, Santos TC et al. 2014b; Gellan gum–hyaluronic acid spongelike hydrogels and cells from adipose tissue synergize promoting neoskin vascularization. ACS Appl Mater Interface 6: 19668–19679. Chung RJ, Hsieh MF, Huang CW et al. 2006; Antimicrobial effects and human gingival biocompatibility of hydroxyapatite sol–gel coatings. J Biomed Mater Res B Appl Biomater 76: 169–178. Doi Y, Horiguchi T, Moriwaki Y et al. 1996; Formation of apatite–collagen complexes. J Biomed Mater Res 31: 43–49. Douglas T, Wlodarczyk M, Pamula E et al. 2014a; Enzymatic mineralization of gellan gum hydrogel for bone tissue-engineering applications and its enhancement by polydopamine. J Tissue Eng Regen Med 8: 906–918. Douglas TE, Messersmith PB, Chasan S et al. 2012; Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase. Macromol Biosci 12: 1077–1089. Douglas TE, Piwowarczyk W, Pamula E, et al. 2014b; Injectable self-gelling composites for bone tissue engineering based on gellan gum hydrogel enriched with different bioglasses. Biomed Mater 9: 045014. Douglas TEL, Krawczyk G, Pamula E et al. 2014c; Generation of composites for bone tissue engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means. J Tissue Eng Regen Med. Douglas TEL, Pilarek M, Kalaszczynska I et al. 2014d; Enrichment of chitosan hydrogels with perfluorodecalin promotes gelation and stem cell vitality. Mater Lett 128: 79–84. Filmon R, Basle MF, Barbier A et al. 2000; Poly(2-hydroxy ethyl methacrylate)-alkaline phosphatase: a composite biomaterial allowing in vitro studies of bisphosphonates on the mineralization process. J Biomater Sci Polym Ed 11: 849–868. Gantar A, Da Silva LP, Oliveira JM et al. 2014; Nanoparticulate bioactive-glass-reinforced gellan–gum hydrogels for bone tissue engineering. Mater Sci Eng C Mater Biol Appl 43: 27–36. Garcia-Fernandez MJ, Brackman G et al. 2013; Antiseptic cyclodextrin-functionalized hydrogels and gauzes for loading and

Copyright © 2015 John Wiley & Sons, Ltd.

delivery of benzalkonium chloride. Biofouling 29: 261–271. Gassling V, Douglas TE, Purcz N et al. 2013a; Magnesium-enhanced enzymatically mineralized platelet-rich fibrin for bone regeneration applications. Biomed Mater 8: 055001. Gassling V, Hedderich J, Acil Y et al. 2013b; Comparison of platelet-rich fibrin and collagen as osteoblast-seeded scaffolds for bone tissue engineering applications. Clin Oral Implants Res 24: 320–328. Giavasis I, Harvey LM, McNeil B. 2000; Gellan gum. Crit Rev Biotechnol 20: 177–211. Grasdalen H, Smidsrod O. 1987; Gelation of gellan gum. Carbohydr Polym 7: 371–393. Habibovic P, Gbureck U, Doillon CJ et al. 2008; Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants. Biomaterials 29: 944–953. Imai K, Nishikawa T, Tanaka A et al. 2010; In vitro new capillary formation with eight metal ions of dental biomaterials. J Oral Tissue Eng 8: 74–79. Ito A, Ojima K, Naito H et al. 2000; Preparation, solubility, and cytocompatibility of zinc-releasing calcium phosphate ceramics. J Biomed Mater Res 50: 178–183. Kawamura H, Ito A, Miyakawa S et al. 2000; Stimulatory effect of zinc-releasing calcium phosphate implant on bone formation in rabbit femora. J Biomed Mater Res 50: 184–190. Kawamura H, Ito A, Muramatsu T et al. 2003; Long-term implantation of zincreleasing calcium phosphate ceramics in rabbit femora. J Biomed Mater Res A 65: 468–474. Kocen R, Gantar A, Novak S. 2014; Viscoelastic properties of gellan gum scaffolds with bioactive glass particles. J Tissue Eng Regen Med 8: 354–355. Lee SK, Silva-Correia J, Caridade SG et al. 2012; Evaluation of different formulations of gellan gum-based hydrogels for tissue engineering of intervertebral disc. J Tissue Eng Regen Med 6: 44. Li X, Sogo Y, Ito A et al. 2009; The optimum zinc content in set calcium phosphate cement for promoting bone formation in vivo. Mater Sci Eng C Mater Biol Appl 29: 969–975. Luo X, Barbieri D, Davison N et al. 2014; Zinc in calcium phosphate mediates bone induction: in vitro and in vivo model. Acta Biomater 10: 477–485. Manda-Guiba MG, Oliveira MB, Mano JF et al. 2012; Gellan gum–hydroxyapatite composite hydrogels for bone tissue engineering. J Tissue Eng Regen Med 6: 15. Miao S, Weng W, Cheng K et al. 2007; In vitro bioactivity and osteoblast-like cell test of zinc-containing fluoridated hydroxyapatite films. J Mater Sci Mater Med 18: 2101–2105. Morris ER, Nishinari K, Rinaudo M. 2012; Gelation of gellan – a review. Food Hydrocolloid 28: 373–411. Mourino V, Cattalini JP, Boccaccini AR. 2012; Metallic ions as therapeutic agents in tissue engineering scaffolds: an overview of their biological applications and strategies

for new developments. J R Soc Interface 9: 401–419. Oliveira JT, Martins L, Picciochi R et al. 2010; Gellan gum: a new biomaterial for cartilage tissue engineering applications. J Biomed Mater Res A 93: 852–863. Osmalek T, Froelich A, Tasarek S. 2014; Application of gellan gum in pharmacy and medicine. Int J Pharm 466: 328–340. Pimenta DR, Silva-Correia J, Caridade SG et al. 2011; Novel gellan gum hydrogels for tissue engineering of intervertebral disc. Int J Artif Organs 34: 703. Pina S, Vieira SI, Rego P et al. 2010; Biological responses of brushite-forming Zn- and ZnSr-substituted β-tricalcium phosphate bone cements. Eur Cell Mater 20: 162–177. Schouten C, Van den Beucken JJ, De Jonge LT et al. 2009; The effect of alkaline phosphatase coated onto titanium alloys on bone responses in rats. Biomaterials 30: 6407–6417. Seo HJ, Cho YE, Kim T et al. 2010; Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract 4: 356–361. Silva-Correia J, Oliveira JM, Caridade SG et al. 2011; Gellan gum-based hydrogels for intervertebral disc tissue-engineering applications. J Tissue Eng Regen Med 5: E97–107. Spoerke ED, Anthony SG, Stupp SI. 2009; Enzyme-directed templating of artificial bone mineral. Adv Mater 21: 425–430. Stanic V, Dimitrijevic S, Antic-Stankovic J et al. 2010; Synthesis, characterization and antimicrobial activity of copper- and zincdoped hydroxyapatite nanopowders. App Surf Sci 256: 6083–6089. Storrie H, Stupp SI 2005; Cellular response to zinc-containing organoapatite: an in vitro study of proliferation, alkaline phosphatase activity and biomineralization. Biomaterials 26: 5492–5499. Thian ES, Konishi T, Kawanobe Y et al. 2013; Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties. J Mater Sci Mater Med 24: 437–445. Verma A, Wahi AK, Pandit JK. 2012; Effect of ++ ++ and Zn in the crosslinking with Ca formation of gellan gum gels. Lat Am J Pharm 31: 815–820. Wang X, Ito A, Sogo Y, et al. 2010; Zinc-containing apatite layers on external fixation rods promoting cell activity. Acta Biomater 6: 962–968. Webster TJ, Ergun C, Doremus RH, et al. 2002; Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. II. Mechanisms of osteoblast adhesion. J Biomed Mater Res 59: 312–317. Yang F, Dong WJ, He FM et al. 2012; Osteoblast response to porous titanium surfaces coated with zinc-substituted hydroxyapatite. Oral Surg Oral Med Oral Pathol Oral Radiol 113: 313–318.

J Tissue Eng Regen Med (2015) DOI: 10.1002/term