Multifunctional magnetic mesoporous bioactive glass scaffolds with a hierarchical pore structure.
ABSTRACT Hyperthermia and local drug delivery have been proposed as potential therapeutic approaches for bone defects resulting from malignant bone tumors. The development of bioactive materials with magnetic and drug delivery properties may potentially meet this target. The aim of this study was to develop a multifunctional mesoporous bioactive glass (MBG) scaffold system for both hyperthermic and local drug delivery applications. To this end iron (Fe)-containing MBG (Fe-MBG) scaffolds with a hierarchical large pores structure (300-500 μm) and fingerprint-like mesopores (4.5 nm) have been prepared. The effects of Fe on the mesopore structure and physiochemical, magnetic, drug delivery and biological properties of MBG scaffolds have been systematically investigated. The results show that the morphology of the mesopores varied from straight channels to curved fingerprint-like channels after incorporation of Fe into MBG scaffolds. The magnetism of MBG scaffolds can be tailored by controlling the Fe content. Furthermore, the incorporation of Fe into mesoporous MBG glass scaffolds enhanced the mitochondrial activity and the expression of bone-related genes (ALP and OCN) in human bone marrow mesenchymal stem cells (BMSC) attached to the scaffolds. The Fe-MBG scaffolds obtained also possessed high specific surface areas and demonstrated sustained drug delivery. Thus Fe-MBG scaffolds are magnetic, degradable and bioactive. The multifunctionality of Fe-MBG scaffolds suggests that there is great potential for their use in the treatment and regeneration of large-bone defects caused by malignant bone tumors through a combination of hyperthermia, local drug delivery and osteoconductivity.
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ABSTRACT: The objective of the study was to examine the effect of alkali ion release, pH control and buffer capacity on the expression of the osteoblastic phenotype. In addition we determined the importance of modifications of the surface of porous bioactive glass (BG) on the activity of rat calvaria osteoblasts in vitro. We found that at a low tissue culture medium (TCM) volume to BG surface area (Vol/SA) ratio, the products of glass corrosion elevated the pH of the TCM to a value that adversely affected cellular activity; thus, the matrix synthesized by the cells was non-mineralized. On the other hand, when the Vol/SA was high and the buffer capacity of the medium was not exceeded, the cells generated a mineralized extracellular matrix. Addressing the second issue, we observed that modification of the composition of the BG surface markedly influenced osteoblast activity. BG that was coated with either a calcium phosphate-rich layer only or a serum protein layer changed the phenotypic characteristics of the osteoblasts. The presence of either of these surfaces lowered the alkaline phosphatase activity of the attached cells; this finding indicated that the osteoblast phenotype was not conserved. However, when the BG was coated with a bilayer of calcium phosphate and serum proteins, the alkaline phosphatase (AP) activity was elevated and the extracellular matrix contained characteristic bone markers. Our findings indicate that the calcium phosphate-rich layer promotes adsorption and concentration of proteins from the TCM, and it is utilized by the osteoblasts to form the mineralized extracellular matrix.Biomaterials 03/1997; 18(4):295-303. · 7.60 Impact Factor
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ABSTRACT: Musculoskeletal tissue, bone and cartilage are under extensive investigation in tissue engineering research. A number of biodegradable and bioresorbable materials, as well as scaffold designs, have been experimentally and/or clinically studied. Ideally, a scaffold should have the following characteristics: (i) three-dimensional and highly porous with an interconnected pore network for cell growth and flow transport of nutrients and metabolic waste; (ii) biocompatible and bioresorbable with a controllable degradation and resorption rate to match cell/tissue growth in vitro and/or in vivo; (iii) suitable surface chemistry for cell attachment, proliferation, and differentiation and (iv) mechanical properties to match those of the tissues at the site of implantation. This paper reviews research on the tissue engineering of bone and cartilage from the polymeric scaffold point of view.Biomaterials 01/2001; 21(24):2529-43. · 7.60 Impact Factor
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ABSTRACT: In a cell culture model of murine osteoblasts three particulate bioactive glasses were evaluated and compared to glass (either borosilicate or soda-lime-silica) particles with respect to their effect on metabolic activity, cell viability, changes in intracellular ion concentrations, proliferation and differentiation. 45S5 Bioglass caused extra- and intracellular alkalinization, a rise in [Ca2+]i and [K+]i, a small plasma membrane hyperpolarization, and an increase in lactate production. Glycolytic activity was also stimulated when cells were not in direct contact with 45S5 Bioglass particles but communicated with them only through the medium. Similarly, raising the pH of culture medium enhanced lactate synthesis. 45S5 Bioglass had no effect on osteoblast viability and, under most conditions, did not affect either proliferation or differentiation. Bioactive glasses 58S and 77S altered neither the ion levels nor enhanced metabolic activity. It is concluded that: (1) some bioactive glasses exhibit well-defined effects in osteoblasts in culture which are accessible to experimentation; (2) 45S5 Bioglass causes marked external and internal alkalinization which is, most likely, responsible for enhanced glycolysis and, hence, cellular ATP production; (3) changes in [H+] could contribute to alternations in concentrations of other intracellular ions; and (4) the rise in [Ca2+]i may influence activities of a number of intracellular enzymes and pathways. It is postulated that the beneficial effect of 45S5 on in vivo bone growth and repair may be due to some extent to alkalinization, which in turn increases collagen synthesis and crosslinking, and hydroxyapatite formation.Biomaterials 02/2001; 22(2):175-85. · 7.60 Impact Factor
This is the author’s version of a work that was submitted/accepted for pub-
lication in the following source:
Wu , Chengtie, Fan, Wei, Zhu, Yufang, Gelinsky, Michael, Chang, Jiang,
Cuniberti, Gianaurelio, Albrecht, Victoria, Friis, Thor, & Xiao, Yin (2011)
Multifunctional magnetic mesoporous bioactive glass scaffolds with a hier-
archical pore structure. Acta Biomaterilia, 7(10), pp. 3563-3572.
This file was downloaded from: http://eprints.qut.edu.au/42571/
c ? Copyright 2011 Elsevier
Notice: Changes introduced as a result of publishing processes such as
copy-editing and formatting may not be reflected in this document. For a
definitive version of this work, please refer to the published source:
Magnetic mesoporous bioactive glass scaffolds with hierarchical pore
structure and multifunction
Chengtie Wu,1,2#* Wei Fan,2# Yufang Zhu,3 Michael Gelinsky,4 Jiang Chang,1 Gianaurelio
Cuniberti,5 Victoria Albrecht,6 Thor Friis,2 Yin Xiao2*
1. Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China.
2. Institute of Health & Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland
3. School of Materials Science and Engineering, University of Shanghai for Science and Technology, 516
Jungong Road, Shanghai, 200093, People’s Republic of China.
4. Centre for Translational Bone, Joint and Soft Tissue Research, Medical Faculty and Hospital, Dresden
University of Technology, Fetscherstr. 74, 01307 Dresden, Germany.
5. Institute for Materials Science and Max Bergmann Center of Biomaterials, Dresden University of Technology,
Budapester Str. 27, D-01069 Dresden, Germany.
6. Leibniz Institute of Polymer Research, Hohe Strasse 6, 01069 Dresden, Germany.
*corresponding-Author Dr. C. Wu., E-mail: firstname.lastname@example.org; Dr Y. Xiao, Email: Yin.Xiao@qut.edu.au
# Co-first authors contributed equally to this work.
Abstract: Hyperthermia and local drug delivery have been proposed the potential therapeutic
approaches for bone defects resulting from malignant bone tumors. Development of bioactive
materials with magnetic and drug-delivery properties may potentially meet this target. The aim of
this study is to develop a multifunctional mesoporous bioactive glass (MBG) scaffold system for
both hyperthermia and local-drug delivery application potentially. For this aim, Iron (Fe) containing
MBG (Fe-MBG) scaffolds with hierarchically large pores (300-500 µm) and fingerprint-like
mesopores (4.5 nm) have been successfully prepared. The effect of Fe on the mesopore structure,
physiochemical, magnetism, drug delivery and biological properties of MBG scaffolds has been
systematically investigated. The results showed that the morphology of the mesopore varied from
straight channels to curved fingerprint-like channels after incorporated parts of Fe into MBG
scaffolds. The magnetism magnitude of MBG scaffolds can be tailored by controlling Fe contents.
Furthermore, the incorporating of Fe into mesoporous MBG glass scaffolds enhanced the
mitochondrial activity and bone-relative gene (ALP and OCN) expression of human bone marrow
mesenchymal stem cells (BMSCs) on the scaffolds. The obtained Fe-MBG scaffolds also possessed
high specific surface areas and sustained drug delivery. Therefore, Fe-MBG scaffolds are magnetic,
degradable and bioactive. The multifunction of Fe-MBG scaffolds indicates that there is a great
potential for Fe-MBG scaffolds to be used for the therapy and regeneration of large-bone defects
caused by malignant bone tumors through the combination of hyperthermia, local drug delivery and
Key words: Magnetic scaffolds; Mesoporous bioactive glasses; Drug delivery; Hyperthermia
Malignant bone tumors are some of the main non-trauma related factors resulting in critical sized
bone loss/defects. The treatment of such bone diseases typically proceed in two stages: the diseased
bone tissue is first surgically removed, resulting in the bone defect, which is then remedied with a
bone graft material . Although such implantations in most cases result in the successful repair of
the defect, the treatment of malignant bone disease and regeneration of the large bone defects still
represents a significant clinical challenge. Hyperthermia therapy by means of biocompatible
magnetic materials has emerged as a potential treatment option against malignant tumors and a
number of recent studies have reported the development of such materials, for example, magnetic
Ca-P ceramics[2,3], bioactive glass-ceramics[4,5] and composites [6,7]. These biomaterials were
designed to be magnetic which, when exposed to a magnetic field, produce heat within the diseased
tissue region; cancer cells are destroyed at temperatures higher than 43oC, whereas the normal cells
can survive these temperatures [1,5,8]. Since blood vessels and the nervous system are poorly
developed in the diseased tissues they tend to be heated more than the surrounding normal tissues.
Hyperthermia treatment is therefore considered an effective treatment option for malignant bone
disease without adverse side effects . Another method that has come under consideration in
treating malignant bone disease is the targeted delivery of drugs through biomaterials, in which the
drug will be efficiently released at the sites of bone disease from loaded biomaterials to either
eradicate the disease or stimulate bone healing [10-12]. Methods such as these are, to some extent,
efficient for the therapy of malignant bone disease but a “one-size-fits-all” method of hyperthermia
or drug-delivery therapy is far from ideal, since a combination of adjunctive treatments modalities,
for example, radio and chemo therapies are often required ; such therapies, however, are nearly
always accompanied by side effects. Hyperthermia treatment is used for killing the local cancer
cells around bone defects; however, the limitation of this method is that if some cancer cells have
migrated to a far location from local bone defects, hyperthermia treatment to local bone defects may
not kill those cancer cells migrated from bone defects. Drug-delivery treatment is able to kill cancer
cells in the whole body system; however, the limitation of drug-delivery treatment is that the side
effects will be accompanied, in which normal tissue cells will be killed if the too much drug dose is
used. Furthermore, how to maintain a sustained drug-delivery from biomaterials is another
challenging issue. We therefore propose a new concept for the treatment of bone disease and bone
regeneration defects: a bioactive material that combines hyperthermia and local drug delivery. This
new concept has the potential to overcome the shortcomings of the simple therapy method; it will
combine the physical (hyperthermia) and chemical (drug delivery) modalities via a multifunctional
scaffold. To satisfy these requirements, the scaffolds have to be biodegradable and bioactive (for
regenerative bone defects); magnetic (for hyperthermia); and possess a special nanostructure with
sustained drug-delivery capacity (for drug therapy). As far as we know there are no traditional
scaffolds suitable for bone tissue regeneration that meet these requirements.
Mesoporous bioglass (MBG), as a new bioactive material, has attracted significant attention in
recent years [14-18]. A significant feature of MBG, compared with non-mesopore bioglass (NBG),
is that it possesses a significantly improved surface area and nanopore volume which is evident by
greatly enhanced bioactivity and degradation. We have recently demonstrated that the MBG has
improved in vitro and in vivo bioactivity, degradation and drug delivery properties, compared to
NBG [19,20]. MBG is a very promising bioactive material with respect to bone regeneration and
drug delivery; however, MBG is not magnetic. Iron (Fe) plays a vital role in the functioning of the
body and the total Fe pool in humans is found in the red blood cell mass, to a lesser extant in the
tissues and a small amount circulating in the plasma . We hypothesized that incorporating
ferrous ions into MBG would make it magnetic and, at the same time, influence its mesopore
structure, physiochemical and biological properties. To our best knowledge, there are no reports
detailing the development of magnetic MBG scaffolds for bone tissue repair, nor are there any
systematic studies of how Fe influences cellular response to MBG scaffolds. Therefore, the aims of
this study were to develop a multifunctional scaffold system which could potentially be used for
hyperthermia and drug-delivery therapy of malignant bone diseases. For this purpose, Fe-containing
MBG scaffolds were prepared and the effect of Fe on the mesopore structure, physiochemical and
biological properties of Fe-MBG scaffolds was systematically investigated.
2. Materials and Methods
2.1 Preparation of porous Fe-MBG scaffolds
Porous Fe-containing mesopore-bioglass (Fe-MBG) scaffolds were prepared by incorporating parts
of Fe (5 and 10%) into MBG using co-templates of nonionic block polymer P123 (EO20-PO70-
EO20) (Sigma Aldrich, Germany) and polyurethane sponges. P123 is used to produce mesoporous
structures (mesopore size: several nanometers) and polyurethane sponges are used to create large
pores (large pore size: several hundred micrometers) as described in our previous publications .
To prepare MBG scaffolds containing of 10% Fe, typically, 6.67 g of P123 (Mw=5800, Sigma
Aldrich, Germany), 11.17 g of tetraethyl orthosilicate (TEOS, 98%, Sigma Aldrich, Germany), 0.78
g of Ca(NO3)2·4H2O (Sigma Aldrich, Germany), 1.12 g of FeCl3 (Sigma Aldrich, Germany), 1.22 g
of triethyl phosphate (TEP, 99.8%, Sigma Aldrich, Germany) and 1.67 g of 0.5 M HCl were
dissolved in 100 g of ethanol (Fe/Ca/P/Si/ = 10/5/5/80, molar ratio, named 10Fe-MBG) and stirred
at room temperature for 1 day. The polyurethane sponges (25ppi) were cleaned and completely
immersed into this solution for 10 min, then transferred to a Petri dish; excess solution was removed
and the remainder allowed evaporating at room temperature for 24 h. This procedure was repeated
for 5 times. Once the samples were completely dry, they were calcined at 700oC for 5 h yielding the
10Fe-MBG scaffolds. MBG scaffolds without Fe (named: 0Fe-MBG) and with 5% Fe (named: 5Fe-
MBG) were prepared by the the same method except for their Fe content. The chemical
composition and the amounts of reagents for 0Fe-MBG, 5Fe-MBG and 10Fe-MBG scaffolds are
listed in Table 1.
2.2 Characterization of Fe-MBG scaffolds
The phase composition, surface morphology, and inner microstructure of the calcined Fe-MBG
scaffolds were analyzed by wide-angle X-ray diffraction (XRD), small-angle XRD, transmission
electron microscopy (TEM) and scanning electron microscopy (SEM) and energy dispersive
spectrometer (EDS). TEM of the samples was performed using a FEI Tecnai 10 electron
microscope (FEI Company, Eindhoven, NL) with a LaB6-source at 100 kV acceleration voltage.
Images were recorded with a Tietz slow scan CCD F224HD TVIPS camera (2k x 2k pixels, pixel
size 24 μm, digitization 16 bit) with an active area of 49 mm × 49 mm. (Tietz Video and Image
Processing Systems GmbH, Gauting, Germany). Brunauer-Emmett-Teller and Barret-Joyner-
Halenda analyses were used to determine the specific surface area, the nano pore size distribution
and the pore volume by N2 adsorption-desorption isotherms.
The porosity of the prepared scaffolds was measured using Archimedes’ principle: scaffolds with a
size of 6×6×6 mm were used for the measurement and water was used as liquid medium. The
porosity (P) was calculated according to the following formulation P = (W2-W1)/(W2-W3) × 100%,
where W1 is the dry weight of the scaffolds, W2 is the weight of scaffolds saturated with water, and
W3 is the weight of scaffolds suspended in water.
2.3 The mechanical strength and magnetic property of Fe-MBG scaffolds
The compressive strength of 10×10×10 mm sized scaffolds were tested using an Instron 5566
computer-controlled universal testing machine (Instron Wolpert, Darmstadt, Germany) at a
crosshead speed of 0.5 mm/min.
To investigate the effect of Fe on the magnetic property, the obtained scaffolds were crushed,
ground and sieved through 300 meshes to obtain powder samples. Magnetic measurements were
carried out with a vibrating-sample magnetometer (LakeShore VSM-7401) at room temperature.
2.4 Ion release and mineralization of Fe-MBG scaffolds
To investigate the ion release and mineralization of Fe-MBG scaffolds, simulated body fluids (SBF)
were prepared according to Kokubo . Fe-MBG scaffolds were soaked in SBF at 37°C for 1, 2
and 3 d, and the ratio of the solution volume to the scaffold mass was 200 mL/g. The concentrations
of Fe2+ (or Fe3+), SiO44-, Ca2+ and PO43- ions in the SBF were determined by atomic emission
spectrometry (Perkin-Elmer Optima 7000DV). After soaking, the surface morphology and
composition of Fe-MBG scaffolds were determined by SEM and EDS.
2.5 Drug loading and release from Fe-MBG scaffolds
Dexamethasone (DEX, Sigma Aldrich, Germany) served as a model drug and was dissolved in
ethanol to a concentration of 0.5 mg/mL. 20 mg of Fe-MBG scaffolds (4 × 4 × 4 mm) were added
to 5 mL of DEX/ethanol solution and soaked for 24 h at room temperature. Fe-MBG scaffolds
loaded with DEX were obtained after drying at 40°C for 24h.
DEX release was evaluated by placing one DEX-loaded Fe-MBG scaffolds into 4 mL of PBS (pH
7.4) at 37 ºC for 3, 9, 24, 48, 96, 168 and 240 h. DEX release was determined by UV analysis (UV
min-1240, Shimadzu, Japan). Then, Fe-MBG scaffolds were crushed and transferred into 5 mL of
PBS. The DEX was solubilized by ultrasonic shaking for 30 min and then the solution was
centrifuged and assayed by UV spectrometry at 240 nm. This was repeated 3 times to ensure the
complete dissolution of the DEX into the PBS. The total amount of DEX loaded in the scaffolds
was the sum of the total release at 240 h and the final release by three readings after ultrasonic
shaking. The accumulative release rate of DEX (%) was calculated with the following equation:
DEX (%) = (total amount of DEX released / total loading amount of DEX in scaffolds) × 100%.
2.6 Morphology and mitochondrial activity of BMSCs on Fe-MBG scaffolds
Isolation and culture of BMSCs was conducted following previously published protocols .
Bone marrow aspirates were obtained from patients (mean age, 65 years) undergoing elective knee
and hip replacement surgery. Informed consent was given by all patients involved and the research
protocol had been approved by the Human Ethics Committees of Queensland University of
Technology and The Prince Charles Hospital.
BMSCs were cultured on 4×4×4 mm scaffolds placed in 96-well culture plates, at an initial density
of 1×105 cells/scaffold. The cells were cultured for 1, 3 and 7 days in DMEM culture medium
(GIBCO) supplemented with 10% FCS, after which the scaffolds were removed from the culture
wells, rinsed in PBS, and then fixed with 1.25% glutaraldehyde, 4% paraformaldehyde, and 4%
sucrose in PBS for 1 hr. The fixative was removed by washing with buffer containing 4% (w/v)
sucrose in PBS and post fixed in 1% osmium tetroxide in PBS followed by CO2 critical-point
drying. The specimens were coated with gold and the morphological characteristics of the attached
cells determined using SEM.
To assess cell mitochondrial activity, an MTT assay was performed  by adding 0.5 mg/mL of
MTT solution (Sigma-Aldrich, Australia) to each scaffold and incubated 37ºC to form formazan
crystals. After 4 h, the media was removed and the formazan solubilized with dimethyl sulfoxide
(DMSO). The absorbance of the formazan-DMSO solution was read at 495 nm on a plate reader.
Results were expressed as the absorbance reading from each well minus the optical density value of
2.7 Reserve transcription and real-time quantitative RT-PCR analysis
The osteogenic differentiation of BMSCs on Fe-MBG scaffolds was further assessed by real-time
quantitative RT-PCR (RT-qPCR) to measure the mRNA expression of ALP and osteocalcin (OCN).
Scaffolds were transferred into 24-well plastic culture plates and a total of 106 BMSCs were placed
onto each scaffold. The cells were incubated at 37ºC in 5% CO2 for 7 days and the medium changed
every 3 days. On day 7, the samples were removed and total RNA isolated using Trizol Reagent®
(Invitrogen) according to the manufacturer’s instructions. Complementary DNA was synthesized
from 1 µg of total RNA using SuperScript III reverse transcriptase (Invitrogen) following the
manufacturer’s instructions. RT-qPCR was performed in 25 µL reaction volume containing 12.5 µL
2X SYBR Green Master Mix (Roche, Castle Hill, NSW, Australia), 2.5 µL each of 10 µM forward
and reverse primers, 2.5 µL of cDNA template diluted 1:10, and 5 µL of RNase free water. All
samples were performed in triplicates and the house keeping gene, 18s rRNA, was used as a control.
The reaction was carried out using ABI Prism 7000 Sequence Detection System (Applied
Biosystems). Melting curve analysis was performed to validate specific amplicon amplification
without genomic DNA contamination. Relative expression levels for each gene were normalized
against the Ct value of the house keeping gene and determined by using the delta Ct method.
2.8 Statistical analysis
All data were expressed as means ± standard deviation (SD) and were analyzed using One-Way
ANOVA with a Post Hoc test. A p-value < 0.05 was considered statistically significant.
3.1. Characterization, mechanical strength and magnetic property of Fe-MBG scaffolds
Small-angle and Wide-angle XRD patterns for the MBG scaffolds with different Fe contents are
shown in Figure 1. There is an obvious diffraction peak around 2θ 1.25 degree for 0Fe-MBG, 5Fe-
MBG and 10Fe-MBG scaffolds (Fig. 1a). Wide-angle XRD analysis showed that none of the three
Fe-MBG species had sharp diffraction peaks (Fig. 1b). Their diffraction patterns had a wide SiO2
peak with low intensity.
All three Fe-MBG scaffold species had a highly porous structure with a similar large pore size
ranging from 300 to 500 µm (Fig. 2a, c and e). EDS analysis showed the main composition of three
species. The ratio of Fe/Ca/Si was 0/16/79, 4.7/8.4/79, and 9.4/5.8/79 for the 0Fe-MBG, 5Fe-MBG
and 10Fe-MBG scaffolds, respectively (Fig. 2b, d and f). The porosity of open pores is 81.6±2.6;
83.3±4.6; and 82.9±2.9 for 0Fe-MBG, 5Fe-MBG and 10Fe-MBG, respectively.
TEM analysis revealed 0Fe-MBG scaffolds to have a well-ordered mesoporous structures with
straight channel (pore size: about 5nm) (Fig. 2a). When 5 and 10% Fe was incorporated, curved
channels with fingerprint-like mesopores could be seen (Fig. 3b and c). The 5Fe-MBG scaffolds
maintained a well-ordered hexagonal structure with a mesopore size of 5 nm (Fig. 3b). Although the
10Fe-MBG scaffolds also had a mesoporeous channel structure, the order of the mesopores seems
appeared more random (Fig. 3c). The results of N2 adsorption–desorption analysis of 10Fe-MBG
scaffolds showed a type of IV isotherm pattern and pore distribution at the 4.5 nm, a typical
characteristic of a mesoporous structure (Fig. 3d). BET analysis showed that the specific surface
area of 10Fe-MBG scaffolds was 268 m2/g.
Incorporating Fe into the MBG scaffolds did not influence their mechanical strength. The
compressive strength is 50±4.7; 48±2.5 and 46±5.4kPa for the 0Fe-MBG, 5Fe-MBG and 10Fe-
MBG scaffolds, respectively.
The magnetization curves of the MBG scaffolds are shown in Figure 4. With the increase of Fe
content, the magnetic magnitude of scaffolds increased. 10Fe-MBG scaffolds had a more obvious
hysterisis loop than the other two scaffolds.
3.2. Ion release and mineralization of Fe-MBG scaffolds
Generally, with the increase of soaking time, the released SiO44- and Ca2+ ions increased; however,
the concentrations of PO43- ions in SBF decreased (Fig. 5). The incorporation of Fe did not change
the release profile of SiO44- ions (Fig. 5a). However, due to Ca being replaced of by Fe, the release
of Ca2+ ions decreased inversely with the increase of Fe contents in MBG scaffolds (Fig. 5b).
Interestingly, as the amount of Fe content increased, the concentrations of PO43- ions in the SBF
decrease (Fig. 5c), the pH of SBF on the other hand fell (Fig. 5d). The detection limit of ICP-AES is
0.143 ppm; however, the Fe ions were not detectible, which indicates that the released Fe from 5Fe-
MBG and 10Fe-MBG scaffolds were below the detection limit.
SEM analysis showed that all three scaffold species had a smooth surface before being soaked in
SBF (Fig. 6a, c and e); however, after soaking, some microparticles were deposited on the surface
of the scaffolds due to the mineralization in SBF (Fig. 6b, d and f). The morphologies of these
microparticles differed significantly between the three scaffold species. Lath-like crystals, ranging
in size from 2–5 µm were formed on 0Fe-MBG scaffolds (Fig. 6b). On the 5Fe-MBG scaffolds
there was a deposition of flake-like particles (Fig. 6d) whereas on the 10Fe-MBG scaffolds very
fine nanosized microparticles were deposited (Fig. 6f). EDS analysis (the inset in Fig. 6b, d and f)
shows that the ratio of Ca/P for 0Fe-MBG, 5Fe-MBG and 10Fe-MBG is 1.70, 1.77 and 1.92,
3.3. Drug release from Fe-MBG scaffolds
All three MBG scaffolds species had a sustained release of DEX (Fig. 7). Incorporation of Fe into
MBG scaffolds did not change the release kinetics of DEX; however, the Fe-containing MBG
scaffolds still maintained a sustained release even after 240 h (The total release is around 80%).
3.4. Attachment, mitochondrial activity and bone-relative gene expression of BMSCs on Fe-MBG
BMSC attachment and morphology on the three scaffolds species were examined by SEM (Fig. 8).
After 7 days in culture, BMSCs spread on the pore walls of the scaffolds. Cells had a close contact
with the substrates by numerous filopodia. The cells grew well on all three scaffolds species (Fig. 9).
There was no obvious difference in the mitochondrial activity between days 1 and 3; however, by
day 7, there was greater cell mitochondrial activity on the 5Fe-MBG and 10Fe-MBG scaffolds
compared to the 0Fe-MBG scaffolds (p<0.05) (Fig. 9). The incorporation of 5% of Fe into MBG
significantly enhanced the bone-related gene expression of OCN and ALP (p<0.05) (Fig. 10).
We have successfully prepared magnetic MBG scaffolds with hierarchically large pores and
fingerprint-like mesopores, and investigated what effects Fe have on mesopore structure,
physiochemistry, magnetism, drug delivery and biological properties. Incorporating Fe into
mesoporous MBG glass scaffolds made them magnetic, as was expected and also significantly
improved cell mitochondrial activity and bone-related gene expression of BMSCs on the scaffolds.
The Fe-MBG the scaffolds were biodegradable, bioactive, magnetic, and at the same time capable
of sustained drug delivery. The multifunction of Fe-MBG scaffolds suggests that they can be used
for the therapy of malignant bone disease by a combination of hyperthermia and local drug delivery,
and for the regeneration of bone defects by virtue of the material’s excellent osteoconductivity (See
Figure 11. the potential application of Fe-MBG scaffolds), in which the magnetic property of
bioactive MBG scaffolds when exposed to a magnetic field, will contribute to the production of the
required heat and temperature to kill cancer cells in bone defects.
We have been able for the first time to prepare magnetic MBG scaffolds using a polymer sponge
method. This is a conventional approach used to prepare porous ceramic scaffolds, and is
particularly useful since it allows for a simple way with which to prepare highly interconnective
pore structure in the scaffolds [25,26]. Our data shows that Fe-MBG scaffolds produced by this
method had high porosity of 83% with large pore sizes ranging from 300 to 500 µm. The porous
properties of these scaffolds greatly benefit tissue ingrowths and nutrient transportation . In
addition, the incorporation of Fe into MBG scaffolds did not influence their overall mechanical
strength, although the mechanical strength of Fe-MBG scaffolds could be described as frail. The
scaffolds were, however, able to maintain their structural integrety when handled for the in vitro
cell biological experiments. In terms of bone tissue engineering applications, the main role of
scaffolds is to provide initial mechanical support for cells, meaning that they must be strong enough
to be handled for this work. In this respect the Fe-MBG scaffolds were able to meet the
requirements as a cell carrier for bone tissue engineering. Our previous studies have shown that the
mechanical strength of mesoporous bioglass scaffolds could be significantly improved by the
modification of silk fibrin  or by 3D-printing preparation method . Therefore, it is believed
that the mechanical strength of Fe-MBG scaffolds could be further improved by using two methods
Mesoporous structure of biomaterials is important for the loading and delivery of drugs [18,28]. Our
previous study has shown that the surface area of MBG without Fe is around 350 m2/g . It is
generally thought that a surface area of 200 to 350 m2/g with a mesopore size of 3 to 5 nm would
provide the right environment to adsorb a range of biomolecules, such as drugs, antibiotics, and
growth factors . 10Fe-MBG scaffolds have high surface area for 268 m2/g, which still falls well
within the optimum surface area range mentioned above, which still falls well within the optimum
surface area range mentioned above. The mesopore size was not affected be the incorporation of Fe,
being approximately 4.5 nm, although it was interesting to note that the incorporation of Fe into the
MBG scaffolds seemed to affect the shape of the mesopores from straight channels to twirled
fingerprint-like channels. These results suggest that the replacement of Ca2+ with Fe3+ may disrupt
the ordered orientation of SiO44- during the self-assembly reaction. The likely reason for this may be
that valence difference between Ca2+ by Fe3+ cause defects of atomic array which manifests itself by
altering the shape of the mesopore channels and surface area. Although the incorporation of 10% of
Fe into MBG scaffolds partially destroyed the ordered mesoporous structure; however, they still
have a high specific surface area, indicating they are capable for adsorbing drugs and subsequently
maintain a sustained release. Earlier studies have incorporated Fe into traditional glass-ceramic and
Ca-P ceramic bulks (not scaffolds) to make them magnetic, but these materials are unsuitable for
drug delivery due to the absence of nanopore structure [2,4,6]. Our results show that the Fe-MBG
scaffolds are well suited for topical drug delivery to bone defect areas and potentially also the
therapy of malignant bone disease.
The magnetic biomaterials are of great value for hyperthermia treatment. In this study, it was
possible to produce magnetism for MBG scaffolds with ferromagnetic properties by replacing Ca2+
with Fe3+; in addition to this, the magnetic magnitude of MBG scaffolds can be tailored by altering
Fe contents, and this is very useful means to control and produce temperature needed to destroy
diseased tissue cells by the process of hyperthermia. The hyperthermia temperature mainly depends
on the magnetic magnitude of materials and external magnetic field. The temperature to kill bone
cancer cells is around 43 oC. It is believed that we can adjust the Fe contents (for different magnetic
magnitude) of MBG scaffolds and external magnetic field to reach this temperature. Therefore, Fe-
MBG scaffolds can combine the hyperthermia therapy and local-drug delivery to treat bone disease,
which may be a more efficient way to improve healing ability compared to a single-therapy method.
The rate of degradation and bioactivity of Fe-MBG scaffolds is an important aspect of their role in
regeneration of bone defects. Fe-MBG scaffolds contain 80% of SiO2 and we found that the
incorporation of Fe into MBG did not influence the release of SiO44- from scaffolds in SBF (Fig. 5).
It is recognized that rates of dissolution play an important role in the degradation of
biomaterials; the Fe-MBG scaffolds we therefore predict will have good degradation properties.
Osteoblast growth and differentiation is influenced by Ca-P mineralization on the surface of
biomaterials which affect their in vivo bone-forming ability [22,30-32]. Although the incorporation
of Fe into MBG modified the morphology of the mineralized Ca-P particles on the surface of
scaffolds in SBF, it did not suppress the Ca-P mineralization, which may indicate that they are
bioactive according to Kokubo’s views . The ionic environment, including ionic concentration
and pH value of SBF, plays an important role to influence the morphology of apatite on bioactive
materials. In this study, the incorporation of Fe into MBG by replacing parts of Ca results in the
decrease of Ca2+ release and pH value in SBF (see Figure 5b and d), which may influence the
morphology of the formed apatite on three scaffolds [33,34]. The ratio of Ca/P for 10Fe-MBG
scaffolds after soaking in SBF is 1.92, which is much higher, compared to that of hydroxyapatite
(1.67). The reason is that the formed Ca-P layer on 10Fe-MBG scaffolds is quite thin (see Figure
6f). When EDS testing was carried out, some Ca and Si signals were also detected from the
substrate of 10Fe-MBG scaffolds themselves, which led to a higher ratio of Ca/P for the formed
Ca-P microparticles. Similar phenomenon was observed for 5Fe-MBG scaffolds (Fig. 6d).
However, for 0Fe-MBG scaffolds, there is a thick Ca-P layer. The ratio of Ca/P for the apatite on
0Fe-MBG scaffolds is about 1.70, which is much close that of hydroxyapatite (1.67).
To further investigate the in vitro bioactivity of Fe-MBG scaffolds, the attachment, mitochondrial
activity and differentiation of BMSCs on the scaffolds were investigated. Our study has found that
no cytotoxicity was observed in the Fe-MBG scaffolds as cell mitochondrial activity was observed
from day 1 to day 7 with significantly increased cell number in the higher Fe scaffold (5Fe-MBG
and 10Fe-MBG) compared with 0Fe-MBG scaffold. Furthermore, 5% of Fe incorporated into MBG
significantly improved both mitochondrial activity and bone-related gene expression (ALP and
OCN), which indicates that a proper amount of Fe will not bring potential cytotoxicity, on the
contrary, it will improve the osteoconductivity of MBG scaffolds. In this study, the incorporation of
Fe into MBG did not influence the large-pore structure of scaffolds, but modified the chemical
composition and further modified the ionic environment of cell culture medium, including the ionic
composition and pH value. Therefore, it is speculated that the ionic composition and pH value are
the direct factors to improve cell viability and differentiation. Previously, Meng et al. found that the
Fe2O3-added poly-lactide/HAp composite film significantly enhanced the proliferation,
differentiation and extracellular matrix secretion of the osteoblast cells . Parsons also found that
the Fe contents play an important role to influence the response of osteoblasts . In our study, it
is speculated that trace amount of Fe3+ released from scaffolds may contribute to the effect of
improved cell response. In addition, pH plays an important role for the cell response [37,38]. In this
study, Fe was incorporated into MBG scaffolds by replacing parts of Ca. The replacing of Ca2+ by
Fe3+ led to a decrease of Ca2+ release, which therefore decreased the pH value of biological solution
(See Figure 5b and d). It can be seen that MBG scaffolds without Fe induced a high pH value (8.0
after 3 days) in biological solution, which is higher than the normal physiological level (pH: 7.4).
However, after incorporating of parts of Fe into MBG scaffolds, Fe-MBG scaffolds induced a closer
pH value to 7.4. Therefore, the beneficial pH environment for Fe-MBG scaffolds may be the other
important factor to benefit the mitochondrial activity and gene expression of BMSCs. Although the
incorporation of Fe into MBG scaffolds decreased the order mesopore structure, they still have a
high surface area compared to traditional non-mesoporous bioglass. From our current results of