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Effect of hydroxyapatite-containing microspheres

embedded into three-dimensional magnesium
phosphate scaffolds on the controlled release
oflysozyme and in vitro biodegradation
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
1 September 2014
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Jongman Lee Abstract: The functionality of porous three-dimensional (3D) magnesium phosphate (MgP)
Hui-suk Yun scaffold was investigated for the development of a novel protein delivery system and biomi-
metic bone tissue engineering scaffold. This enhancement can be achieved by incorporation of
Powder and Ceramics Division,
Korea Institute of Materials Science, hydroxyapatite (HA)-containing polymeric microspheres (MSs) into a bulk MgP matrix, and a
Changwon, Republic of Korea paste-extruding deposition (PED) system. In this work, the amount of MS and HA was precisely
controlled when manufacturing MS-embedded MgP (MS/MgP) composite scaffolds. The main
influence was researched in terms of in vitro lysozyme-release, in vitro biodegradation, mechani-
cal properties, and in vitro calcification. The controlled release of lysozyme was indicated, while
showing graded release patterns according to HA content. The composite scaffolds degraded
gradually with MS content and degradation time. Due to the effect of HA inclusion, the higher
HA-containing MS/MgP scaffolds could, not only delay the biodegradation process but also,
compensate for the possible loss of mechanical properties. In this regard, it is reasonable to
confirm the inverse relationship between biodegradation and corresponding compressive proper-
ties. In order to encourage bioactivity and osteoconductivity, the MS/MgP composite scaffolds
were subjected to simulated body fluid treatment. Calcium deposition was, in turn, improved
with increasing MS and HA content over time. This quantitative result was also proved using
morphological and elemental analysis. In summary, a significant transformation of a monolithic
MgP scaffold was directed toward a multifunctional bone tissue engineering scaffold equipped
with controlled protein delivery, biodegradability, and bioactivity.
Keywords: protein delivery, bone tissue engineering

Introduction
The usefulness of microspheres (MSs) has been extended to combination with a
continuous bulk matrix: solid polymer, hydrogel, and calcium phosphate cement (CPC).1
To this end, they can affect physicochemical and biological characteristics, in terms
of porosity,2 mechanical properties,2 biodegradation,3 the release kinetics of bioactive
agents,4 and bioactivity.3 One of the greatest advantages of introducing MS into a bulk
Correspondence: Hui-suk Yun matrix is to allow final composite scaffolds controlled delivery of biomolecules in a spa-
Powder and Ceramics Division, Korea tiotemporal manner. This is mainly because the direct incorporation of bioactive agents
Institute of Materials Science (KIMS), 797
Changwondaero, Seongsangu, Changwon into bulk scaffolds might be vulnerable to denaturation when exposed to harsh prepara-
642-831, Republic of Korea tion conditions (organic solvent, acidic byproduct, hydrophobicity, and so on).5
Tel +82 55 280 3351
Fax +82 55 280 3392
First, a composite system of MS-embedding solid polymers was developed for the
Email [email protected] sustained release of biomolecules and promoted tissue regeneration compared with

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MS-free scaffolds.3,6 Porous nano-hydroxyapatite (HA)/ between nanosized HA and the lysozyme protein. For this
collagen/poly(L-lactic acid) (PLLA) composite scaffolds reason, we combined the MgP scaffold with HA-containing
containing chitosan MSs were prepared using a thermally polymeric MSs to produce the 3D bone tissue engineering
induced phase separation method.3 The compressive proper- composite scaffold.
ties increased as the chitosan MS content increased, but the In the present work, our aim was to develop MS-
porosity decreased. The in vitro biodegradation rate increased embedding MgP (MS/MgP) composite scaffolds that could
with the enhancement of chitosan MSs, which in turn led to contribute to in vitro lysozyme-release characteristics, in
temporally controlled release of synthetic peptide. Second, vitro biodegradation/corresponding mechanical properties,
a composite system of MS-embedding hydrogel was devised and in vitro calcification, in contrast to the bare MgP scaf-
to serve as a controlled drug delivery carrier,4 reinforcement fold. For this purpose, MS/MgP composite scaffolds were
component,7 and cell delivery vehicle.8 The biodegrad- prepared into two groups: (1) controlled MS content in
able gelatin MS/hydrogel composites were prepared to MS/MgP scaffolds, ranging from 0% to 20% (w/w), and (2)
accomplish a simultaneous delivery of transforming growth controlled HA content in MSs, ranging from 0% to 55%.
factor-1 (TGF-1) and chondrocytes, respectively, for car- A synergistic combination of MgP scaffold and HA-
tilage tissue engineering applications.4 As a result, a much containing polymeric MSs will be useful to create an efficient
slower release of TGF-1 to the gelatin MS-encapsulating protein delivery carrier and bioactive physical platform for
hydrogel composites was seen over the course of 28 days. bone tissue engineering.
Injectable alginate hydrogel composites combined with
-tricalcium phosphate (-TCP) MSs were invented to over- Materials and methods
come the lack of initial mechanical strength of the hydrogel Preparation of MSs and MgP powder
constructs for bone tissue engineering.7 Third, a composite The development of gelatin/chitosan MSs, consisting of
system of MS-embedding CPC was researched extensively nanosized HA (Sigma-Aldrich Corp., St Louis, MO, USA),
to solve the major drawbacks of CPC: slow degradation rate was reported in our previous publication.13 Briefly, 2.5%
attributed to a lack of macroporosity and poor drug-release gelatin type B/chitosan with a weight ratio of 2 to 1 (Sigma-
capacity.1 In this regard, many types of biodegradable poly- Aldrich Corp.) was dissolved in 50 mL of 0.1 M acetic acid
meric MSs were homogeneously incorporated into bone (Sigma-Aldrich Corp.). Different amounts of HA (0%, 1.5%,
cements, not only to facilitate in vitro/in vivo biodegradation and 3.0% [w/v]) were added to this solution and ultrasoni-
and the resultant formation of well-interconnected macropo- cated for 5 minutes, for homogeneous dispersion of the HA.
rous structures, but also, to obtain the controlled release of MSs with no HA were made for comparison following the
bioactive agents for a prolonged period of time, without an same protocol. In this work, the theoretical percentage of HA
initial drug burst.911 in the total solid content was 38 wt% for a mixture containing
In our previous studies, we developed a novel room- 1.5% (w/v) HA and was 55 wt% for 3.0% (w/v) HA. For this
temperature process of three-dimensional (3D) magnesium reason, the final composite MSs with different HA content
phosphate (MgP) scaffold fabrication, using a paste-extruding (0%, 38%, and 55%) were denoted as MS (0% HA), MS
deposition (PED) system.12 This technique enabled us to (38% HA), and MS (55% HA), respectively. The mixture was
directly blend lysozyme, as a model bioactive substance, added dropwise into 5% cellulose acetate butyrate (Eastman
into MgP powder, to introduce homogeneous distribution Chemical Co., Kingsport, TN, USA) dissolved in 200 mL of
in the scaffold. This was completely ascribed to a simple n-butyl acetate (Samchun Pure Chemical Co., Ltd., Seoul,
cementation process at room temperature, instead of the Republic of Korea) while stirring at 200 rpm for 30 minutes.
typical sintering process at high temperature. Both the drug- The gelatin/chitosan MSs were subsequently cross-linked
loading efficiency and in vitro release performance were, in by adding glutaraldehyde (Merck & Co., Inc., Whitehouse
turn, substantially enhanced compared with physical adsorp- Station, NJ, USA) to the emulsion at a final concentration of
tion using lysozyme-containing solution. Nevertheless, we 0.25%, and stirred overnight. The composite MSs were then
wished to develop a more advanced delivery system with a washed with acetone, rinsed in deionized (DI) water, and
high efficacy. We recently investigated the manufacture of then collected using sieves with 150 m and 250 m pores.
HA-containing polymeric MSs as a novel protein delivery The residual glutaraldehyde from the cross-linking process
carrier.13 High protein-loading capacity and controlled release was neutralized by placing the MSs in 2.0% sodium bisulfide
kinetics were achieved by strong electrostatic interactions for 1 day and then washing with DI water. The washed MSs

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Dovepress Microsphere/magnesium phosphate composite scaffolds

were lyophilized for 1 day and stored in a desiccator until apparent porosity of MS/MgP scaffolds (n=5) was calculated
further use. according to the following equation:
MgP powder (farringtonite [Mg 3(PO 4) 2]) was pre-
pared following the methods described in our previous Porosity = [1- (Dscaffold/Dbulk)] 100, (1)
publication.12 Briefly, Mg(OH)2 (Junsei Chemical Co., Ltd.,
where Dscaffold means density of scaffold and Dbulk is density
Tokyo, Japan) and H3PO4 (DC Chemical Co., Ltd., Seoul,
of bulk.
Korea) were reacted with a molar ratio of 3 M to 2 M. Then,
2M of H3PO4 was added to a 3 M of Mg(OH)2 suspension in
Morphological analysis of MS/MgP
a dropwise manner. The solution was mixed uniformly for
10hours and left for 24 hours to age. The precipitate was
composite scaffold
For the morphological analysis, MS/MgP composite scaf-
filtered and dried at 80C for 2 days and heat-treated at 850C
folds were first sputter-coated with gold to minimize sample
for 6hours. The final product was ground in a planetary ball
charging problems and evaluated by scanning electron micro-
mill for 2 hours at a speed of 250 rpm.
scope (SEM) (JSM-6610LV; JEOL Ltd., Tokyo, Japan) at
Preparation of MS/MgP composite an operating voltage of 15 kV. Surface and cross-sectional
SEM micrographs were taken for each MS/MgP composite
scaffold
scaffold, at magnifications of 50 and 100.
In this work, the preparation of MS/MgP composite scaf-
folds could be categorized into two groups: (1) controlled
MS content in MS/MgP scaffolds and (2) controlled HA
Water absorption
For this test, the various MS (55% HA) content samples,
content in MS. The MS content ranged from 0% to 20%
ranging from 0% to 20% (w/w), were selected to constitute
(w/w) in the 55% HA-containing MS/MgP scaffolds; the
MS/MgP composite scaffolds. The water absorption of the
HA content ranged from 0% to 55% in the 10% MS/MgP
MS/MgP composite scaffolds was determined by measuring
scaffolds. The HA-containing composite microspheres and
hydrated weight in DI water at 37C for 3 hours, with gentle
MgP powder were mixed together using a 1% hydroxypropyl
shaking (n=5). MS/MgP scaffolds were then carefully patted
methyl cellulose solution (HPMC) (Sigma-Aldrich Corp.),
with tissue to remove excess water and weighed to measure
with a powder/liquid ratio of 3 to 23 (wt:vol). A uniform
the hydrated weight (Wh). The percentage increase in water
MS/MgP green paste was formed for the PED system. In
absorption can be calculated in contrast to the dried weight
order to visualize the MSs in the MS/MgP composite scaf-
(Wd) of the MS/MgP scaffolds, according to the following
folds, as shown in Figure 1, MS (55% HA) was stained
equation:
with 0.4% trypan blue solution (Gibco; Life Technologies,
Carlsbad, CA, USA), washed in DI water vigorously, and
Water absorption (%) = ([Wh - Wd]/Wh) 100 (2)
lyophilized for 1 day, prior to mixing with MgP powder. The
MS/MgP paste, housed in a syringe, was mounted to a gantry
robotic deposition apparatus, and equipped with specially- Compressive properties
altered systems, such as an actuator, to control the position Based on the MS/MgP scaffold fabrication method described
of the deposition nozzle for the fabrication of 3D structures. above, MS/MgP composite scaffolds with different MS- and
A nozzle size of 21G was used to fabricate the 3D MS/MgP HA-content were produced, while controlling the strut dis-
scaffolds (10104 mm) with a center-to-center distance of tance of 1.0 mm and nozzle size of 21G. The dimension of
1 mm. The MS/MgP scaffold green bodies were dried at room the MS/MgP scaffolds was 10104 mm. The compressive
temperature, followed by immersion in 3.5 M diammonium strength and modulus were measured using a uniaxial testing
hydrogen phosphate (DAHP) (Junsei Chemical Co., Ltd.) machine, with a 2 kN load cell at a cross-head rate of 1mm/min
for 1 day, with gentle shaking to induce a cement reaction. (RB Model 302 ML; R&B Inc., Daejon, Korea) (n=5).
They were then washed in DI water three times and dried
completely by lyophilization. The MgP scaffold without the In vitro biodegradation
inclusion of MSs was also prepared for comparison. The and compressive properties
final MS/MgP scaffolds were observed under a stereomicro- MS/MgP composite scaffolds, with a dimension of 1010
scope (SMZ 1,500; Nikon Corp., Tokyo, Japan) to examine 4 mm, were measured for dried weight prior to beginning
the morphology, strut thickness, and pore size (n=5). The this test. In order to provide the accelerated conditions for an

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A
1 2 3 4 5

B MgP 2% MS/MgP 5% MS/MgP

10% MS/MgP 20% MS/MgP

Figure 1 Gross images of MS/MgP composite scaffolds.

Notes: Gross images of MS/MgP composite scaffolds (A): (1) MgP, (2) 2% MS/MgP, (3) 5% MS/MgP, (4) 10% MS/MgP, and (5) 20% MS/MgP. Closer observation of MS/MgP
composite scaffolds, via a stereomicroscope (3 magnification) (B).
Abbreviations: MgP, magnesium phosphate; MS, microsphere.

in vitro biodegradation test, 10 mL of collagenase solution d ifferent HA content (0% to 55%) was incubated overnight
(50g/mL) (Sigma-Aldrich Corp.) containing 0.1% NaN3 with 2 mL of lysozyme solution in phosphate-buffered
(Sigma-Aldrich Corp.) was transferred, to submerge the MS/ saline (PBS) (0.5 mg/mL). The supernatant was then
MgP scaffolds fully during the whole incubation time. Sam- collected to measure ultraviolet (UV) absorbance at
ples were placed in an incubator temperature-controlled at 280 nm, and the amount of lysozyme loaded into the
37C, while providing gentle shaking. At each predetermined composite MSs was calculated based on a standard curve,
time (days 5, 15, and 30), samples were collected, washed prepared using a series of lysozyme concentrations. The
with DI water three times, and lyophilized to obtain the dried composite MSs were then washed once with DI water to
weight. The percentage of weight reduction was then calcu- eliminate nonspecifically bound lysozyme and lyophilized,
lated in comparison with the initial dried weight (n=5). The prior to blending with MgP powder. Subsequently, 10%
corresponding compressive properties at each biodegradation MS/MgP composite scaffolds were made following the
time (n=5) were evaluated as well, in terms of compressive same method shown above. For comparison, a direct
strength and modulus, to investigate the relationship between addition of lysozyme into MgP scaffold was also prepared
in vitro biodegradation and compressive properties. without using MSs as a lysozyme carrier. The amount of
lysozyme for this sample was the same as that loaded into
In vitro lysozyme-release kinetics MS (55% HA), which was the largest lysozyme content
In order to investigate the release kinetics of lysozyme in all the MS samples with different HA content. Briefly,
from the MS/MgP composite scaffolds, 10% MS with lysozyme, dissolved in 1% HPMC solution, was uniformly

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Dovepress Microsphere/magnesium phosphate composite scaffolds

blended with MgP powder to achieve a direct incorpora- significant. A value of P0.01 or P0.05 was accepted as
tion of lysozyme inside MgP scaffold. After lyophilizing statistically significant.
the lysozyme-loading scaffolds, the dried weight was
measured to calculate the lysozyme-loading content per Results and discussion
scaffold; 0.37g for MgP, 0.27g for MS (0% HA)/MgP, Microscopic characterization of MS/MgP
0.32g for MS (38% HA)/MgP, and 0.32g for MS (55% composite scaffolds
HA)/MgP (n=5). The lysozyme-release test began by plac- HA-containing composite MS was prepared according to
ing MS/MgP composite MSs into 5 mL of PBS containing the preparation method described above. As described in our
0.1% NaN3 at 37C, with gentle shaking (150 rpm). At each previous publication, the mean diameters for the composite
predetermined time, a sample of the medium was collected MS in the dried state increased slightly with increasing HA
and replaced with an equal volume of fresh medium. The content, from 8511 m to 9512 m to 10113 m.13
lysozyme content was analyzed quantitatively using a The gross images of MS/MgP composite scaffolds are
micro BCA protein assay kit (Thermo Fisher Scientific, shown in Figure 1A. The dimension of the MS/MgP scaffolds
Inc., Waltham, MA, USA) according to the manufacturers (10104 mm) was well maintained without any expansion
instructions (n=5). or shrinkage during the whole manufacturing process. Due
to the advantage of the PED system, the structures could be
In vitro calcification accurately controlled so as to fabricate well-interconnected
The capacity of MS/MgP composite scaffolds (10104 porous 3D scaffolds. A gradual increase in blue color could
mm) to absorb calcium ions was estimated by adding each be seen while adding the blue-stained MSs into MS/MgP
MS/MgP scaffold to 10 mL of simulated body fluid (SBF) composite scaffolds ranging from 0% to 20% (w/w). This
and incubating at 37C for 5, 10, and 20 days, with gentle was more evident when observing the MS/MgP composite
shaking. The SBF was prepared according to previously scaffolds under a stereomicroscope (Figure 1B). Moreover,
reported studies.14 At each predetermined time (day 5, 10, it seemed that the surface of the composite scaffolds became
and 20), SBF was collected and replaced with fresh fluid. rougher as more MS content was added to MS/MgP scaffolds.
The calcium ion content deposited on the MS/MgP com- The strut thickness and pore size of all MS/MgP composite
posite scaffolds was then determined qualitatively using a scaffolds were similar, at 540580 m and 420440 m,
commercially available assay kit (QuantiChrom Calcium respectively (n=5). In addition, the apparent porosity was
Assay Kit, BioAssay Systems, Hayward, CA, USA) (n=5). found to be 50%52% (n=5).
The initial calcium ion concentration of SBF was com- In the SEM micrographs (Figure 2), the existence of MS
pared with the retrieved SBF solution at each time noted, was clearly seen, either in the core or on the surface of the
to calculate the calcium ion deposition. In brief, 5 L of struts. As was shown in Figure 1, more MSs could be found
retrieved samples were reacted with 200 L of working in the MS/MgP composite scaffolds while increasing MS
solution and incubated for 3 minutes at room temperature, content from 2% to 20% (w/w). As a result, the 20% MS/MgP
followed by measurement of UV absorbance at 612 nm, scaffold exhibited the highest distribution of MS content,
using a UV-Visible spectrophotometer (UVmini-1240; whereas others contained relatively few MSs.
Shimadzu Corp., Kyoto, Japan). After completing the
calcium ion deposition test in SBF, the samples at day 20 Water absorption
were washed carefully with DI water and dried thoroughly The capacity of water absorption (%) by the MS/MgP
to carry out a morphological and elemental analysis by scaffolds was explained in Figure 3. Bare MgP scaffold
SEM and energy dispersive spectroscopy (EDS) (Oxford showed the lowest water absorption, of 39%, after soak-
Instruments, Abingdon, UK), respectively. ing in DI water for 3 hours. However, the water uptake
increased gradually with increasing MS content from 2%
Statistics to 20%. The 10% and 20% MS/MgP composite scaffolds
All quantitative results were expressed as means standard showed a dramatic increase in water uptake, to 48% and
deviation for number n of samples analyzed. Statistical 53%, respectively.
analysis was carried out using one-way analysis of variance According to our previous work on HA-containing com-
(ANOVA), followed by the Tukey test for post hoc mul- posite MSs,13 the water content (%) of the composite MSs
ticomparisons to determine whether the differences were varied from 64.5% to 81.1%, depending on the HA content

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MgP 2% MS/MgP 5% MS/MgP 10% MS/MgP 20% MS/MgP

Figure 2 SEM measurement of MS/MgP composite scaffolds.

Notes: The surface (top row) and cross-sectional (middle and bottom row) micrographs were viewed at magnifications of 50 and 100.
Abbreviations: MgP, magnesium phosphate; MS, microsphere; SEM, scanning electron microscope.

embedded in polymeric MSs. In this work, the experiments increasing amount of hydrophilic composite MSs enveloped
were mostly conducted using 55% HA-containing MSs. This in the MS/MgP composite scaffolds.
MS has the lowest water content (%), of 64.5%, but still had
excellent hydrophilic capacity. Due to the significant influ- Compressive properties
ence of MSs, it was possible to enhance the hydrophilic prop- The compressive properties of MS/MgP composite scaffolds
erties of bare MgP scaffold by simple incorporation of MS are depicted in Figure 4. The compressive properties of MgP
into the MgP matrix. Thus, 10% and 20% MS/MgP scaffolds scaffolds are indicated using a dotted line for comparison.
were able to achieve improvement in water absorption (%) The HA-containing MS/MgP composite scaffolds maintained
relative to the MgP scaffold (**P0.01). The proportional the compressive strength and modulus, both similar to the
increase in water absorption (%) could be attributed to the level of MgP scaffold throughout all MS content samples
(2% to 20%). However, 0% HA-containing MS/MgP scaf-
folds resulted in a gradual decrease in compressive strength,
60
which finally reached its lowest, at 20% MS/MgP scaffolds
**
(P0.01). Regarding the compressive modulus, most of
the MS/MgP composite scaffolds exhibited similar values
Water absorption (%)

50 ** to the MgP scaffold, except for 20% MS/MgP scaffolds. In

this case, a significant reduction was only seen in the 0%
HA-containing MS/MgP scaffold (P0.05). The inclusion
of HA into MS was therefore advantageous to the MS/MgP
40
composite scaffolds because the HA addition would be able
to preserve the original mechanical characteristics of the
MgP scaffold and also, prevent a substantial decrease in
compressive properties.
30
MgP S/Mg
P
S/Mg
P
MS/M
gP
MS/M
gP
2% M 5% M 10% 20%
In vitro biodegradation and compressive
Figure 3 The water absorption (%) of MS/MgP composite scaffolds was evaluated
by comparing the weight differences between dried and hydrated samples.
properties
Notes: MS (55% HA) was used to fabricate 0% to 20% MS/MgP composite scaffolds. The in vitro biodegradation of MS/MgP composite scaf-
A significant increase in water absorption (%) was found in 10% and 20% MS/MgP
folds were evaluated in two different aspects: the effect of
scaffolds in comparison with MgP scaffolds (**P0.01).
Abbreviations: HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere. MS content in MS/MgP scaffolds (Figure 5A) and of HA

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Dovepress Microsphere/magnesium phosphate composite scaffolds

A B
14 50
MS (0% HA)
MS (38% HA) MS (0% HA)
12 MS (55% HA) MS (38% HA)

Compressive modulus
Compressive strength
40 MS (55% HA)
10 ** **
**
* * *
MgP 30
8 MgP
(MPa)

(MPa)
6
20

4
10
2

0 0
2% MS/MgP 5% MS/MgP 10% MS/MgP 20% MS/MgP 2% MS/MgP 5% MS/MgP 10% MS/MgP 20% MS/MgP

Figure 4 Compressive properties of MS/MgP composite scaffolds were tested to investigate the effect of MS content in MS/MgP scaffolds and of HA content in MS on
compressive strength (A) and compressive modulus (B).
Notes: Statistical significance was evaluated in contrast to 0% HA-containing MS/MgP scaffolds at each group (*P0.05 or **P0.01).
Abbreviations: HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere.

content in MS (Figure 5B). The steady increase in the in vitro to decrease after being biodegraded over time (Figure 6A).
biodegradation was monitored over time. This meant that all Contrary to the in vitro biodegradation result (Figure 5), the
MS/MgP composite scaffolds would be degraded gradually in more the MS/MgP composite scaffolds were degraded, the
proportion to the incubation time. The in vitro biodegradation fewer compressive properties were obtained as a function of
was facilitated with increasing MS content, from 0% to 20%. biodegradation time. For this reason, the lowest compressive
The greatest weight reduction occurred with the 20% MS/ strength was found with 20% MS/MgP composite scaffolds
MgP composite scaffolds throughout the whole incubation from day 5 to day 30. Due to the superior biodegradability
time (day 5 and day 15 P,0.01; day 30 P,0.05). With regard of the HA-containing 10% MS/MgP composite scaffolds,
to the influence of HA content in MS, a substantial increase significant decrease in compressive properties was found
in biodegradation (%) was shown with the reduction of HA with 0% and 38% HA-containing 10% MS/MgP scaffolds
content from 55% to 0% (P0.01). (Figure 6B). However, the 55% HA-containing 10% MS/
The corresponding compressive properties at each bio- MgP scaffolds were able to prevent this significant loss of
degradation time are shown in Figure 6, and this revealed a mechanical properties to some extent.
close relationship to the in vitro biodegradation. The com- In Figure 5, the in vitro biodegradation pattern of MS/
pressive strength of MS/MgP composite scaffolds began MgP composite scaffolds was highly associated with MS

A B
30 30 ** Day 5
Day 5
Day 15 ** Day 15
Day 30 25
** Day 30
25
**
Biodegradation (%)

Biodegradation (%)

**
20 * 20
**
**
15 15
** **

10 10

5 5

0 0
MgP gP gP gP gP gP gP gP
MS/M MS/M MS/M MS/M HA)/M HA)/M HA)/M
2% 5% 10% 20% (0% (38% (55%
MS MS MS

Figure 5 In vitro biodegradation of MS/MgP scaffolds was carried out by immersion in enzymatic solution for 30 days.
Notes: The effect of MS content in MS/MgP scaffolds (A) and of HA content in MS (B) was investigated. MS content was controlled from 0% to 20% in 55% HA-containing
MS/MgP scaffolds; HA content was controlled from 0% to 55% in 10% MS/MgP scaffolds. Statistical significance was evaluated in contrast to MgP scaffolds (A) and MS (55%
HA)/MgP scaffolds (B) (*P0.05 or **P0.01).
Abbreviations: HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere.

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A
0 day
50
5 days 0 day
14
15 days 5 days

Compressive modulus (MPa)

Compressive strength (MPa)

30 days 15 days
12 40
30 days
*
10 **
* **
30
8

6 20
*
4
10
2

0 0
MgP gP gP gP gP MgP gP gP gP gP
2% MS/M 5% MS/M MS/M MS/M 2% M
S/M
5% M
S/M MS/M MS/M
10% 20% 10% 20%
B
14 50
0 day 0 day
5 days 5 days

Compressive modulus (MPa)

Compressive strength (MPa)

12
15 days 15 days
40
30 days 30 days
10
**
** **
30 **
8

**
6
20

4
10
2

0 0
MS (0% HA)/MgP MS (38% HA)/MgP MS (55% HA)/MgP MS (0% HA)/MgP MS (38% HA)/MgP MS (55% HA)/MgP

Figure 6 The corresponding compressive properties at each biodegradation time were evaluated with respect to compressive strength and modulus.
Notes: The effect of MS content in MS/MgP scaffolds (A) and of HA content in MS (B) was investigated. MS content was controlled from 0% to 20% in 55% HA-containing
MS/MgP scaffolds; HA content was controlled from 0% to 55% in 10% MS/MgP scaffolds. Statistical significance was evaluated in contrast to scaffolds at day 30 (*P0.05 or
**P0.01).
Abbreviations: HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere.

content (%), HA content (%), and degradation time (day). subsequently lead to a gradual decrease in compressive
In this work, we recognized that MS/MgP composite scaf- strength with MS content and degradation time, which is
folds would be susceptible to degrade in enzymatic solutions very consistent with our results as shown in Figures 5 and 6.
because the MSs were mainly constituted of biodegradable As well, the bulk erosion of gelatin MSs began after 3weeks
natural polymers, such as gelatin and chitosan. As a result, degradation so that many macropores with interconnectivity
the largest weight reduction took place in the 20% MS/ was spread out everywhere.
MgP composite scaffolds at day 30. The biodegradation of Given the influence of HA content in MS, the inclusion
MS-embedding bioceramic scaffolds has been extensively of HA into the MSs was able to, not only retard the bio-
studied under in vitro and in vivo circumstances.9,10 In most degradation process but also, compensate for the potential
cases, the biodegradable polymeric MS was impregnated into loss of compressive properties in a sustained manner. In
bioceramic matrix in a certain ratio and kept under physi- comparison with the biodegradation (%) at day 30, 0%
ological conditions for a prolonged period of time. Then, HA-containing 10% MS/MgP scaffold was 27.5%, which
the result was usually evaluated in terms of the morphology, was twice as large as 13.2% (55% HA-containing 10% MS/
porosity (%), weight loss (%), mechanical properties, and MgP scaffold; Figure 5B). However, the corresponding
so on. For example, a composite scaffold of gelatin MS/ compressive strength of these two samples was measured as
CPC was prepared to create a macroporous structure after 0.3 MPa and 4.5 MPa, respectively. It is therefore straight-
being degraded in PBS for 5 weeks.15 A higher porosity was forward that the improved compressive properties of the
obtained with the degradation of gelatin MS. This could 55% HA-containing MS/MgP composite scaffolds were

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Dovepress Microsphere/magnesium phosphate composite scaffolds

mostly attributed to the significant effect of HA inclusion. MS with different HA contents was: 810 g for MS
In order to reinforce the mechanical properties of bone tissue (0% HA), 889 g for MS (38% HA), and 891 g for MS
engineering scaffolds, HA has been typically incorporated (55% HA). Second, the final lysozyme-loading content
into a bulk polymeric phase, with or without surface modi- of the 10% MS/MgP composite scaffolds was calculated
fication: PLLA,16 poly (-caprolactone),17 and chitosan.18 In based on the dried weight of each sample: 166 g for
most cases, the compressive properties of HA-containing MgP scaffold, 109 g for MS (0% HA)/MgP scaffold, 140
composite scaffolds would improve with the increasing g for MS (38% HA)/MgP scaffold, and 140 g for MS
weight ratio and remain stabilized until a certain point. This (55% HA)/MgP scaffold. In Figure 7A, the MgP scaffold,
is mainly ascribed to the enhanced interaction between HA which had direct blending with lysozyme, showed an
and the bulk matrix and good dispersion of HA inside the initial burst until 7days and then continued to retain the
matrix.17 Furthermore, it was reported that the electrostatic highest release of lysozyme until 30 days. On the other
interactions of gelatin and chitosan with HA nanoparticles hand, the MS/MgP composite scaffolds showed a con-
could slow down the in vitro biodegradation and mechani- trolled release rate without any serious loss and showed
cal properties.18,19 As with the previous studies, we also graded lysozyme-release patterns in accordance with HA
confirmed the inverse relationship between the in vitro content. The lysozyme release kinetics of composite scaf-
biodegradation and corresponding compressive properties folds is shown in order of MS (38% HA)/MgP and MS
of MS/MgP composite scaffolds. The critical importance of (55% HA)/MgP. A slower release patten of MS (55%
the HA addition into MSs was emphasized, so as to prevent HA)/MgP is attributed to the strong affinity of HA to
a severe reduction of compressive properties with in vitro lysozyme as depicted in Figure 7A.
biodegradation time. In our previous study, an HA-containing gelatin/chi-
tosan MS was developed as an efficient protein delivery
In vitro lysozyme-release kinetics carrier that not only enables the loading of high quantities
For this experiment, 10% MSs with different HA content of lysozyme, but also, maintains a sustained release without
were homogeneously embedded into MgP scaffolds to an initial burst.13 The HA effect on lysozyme delivery was
investigate the effect of HA on the in vitro lysozyme- enhanced with increases in HA content from 38% to 55%.
release kinetics. The in vitro release pattern was monitored This outstanding feature could be explained by the increase
for 30days and analyzed in terms of cumulative release in specific surface areas and strong electrostatic interac-
amount (g) (Figure7A) and cumulative release percent- tions of HA-containing composite MSs. For more advanced
age (%) (Figure 7B). The lysozyme-loading content of applications, we endeavored to combine the novel MS with
MS and MS/MgP composite scaffolds could be obtained MgP scaffolds, to allow for the higher loading capacity and
as follows. First, the lysozyme-loading content of 10% prolonged release of lysozyme in a controlled manner. As

A MgP
B
150 MS (0% HA)/MgP 100 MgP
Cumulative release of lysozyme (g)

Cumulative release of lysozyme (%)

MS (38% HA)/MgP MS (0% HA)/MgP

MS (55% HA)/MgP MS (38% HA)/MgP
80 MS (55% HA)/MgP

100
60

40
50

20

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

Days Days

Figure 7 In vitro lysozyme-release kinetics of MS/MgP composite scaffolds was monitored for 30 days and expressed in two different aspects: (A) cumulative release amount
(g) and (B) cumulative release percentage (%) against lysozyme loading amount.
Abbreviations: HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere.

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Table 1 Quantitative analysis of in vitro calcium deposition was carried out, in terms of MS content, in 55% HA-containing MS/MgP
scaffolds
Ca deposition (g/mL) MgP 2% MS/MgP 5% MS/MgP 10% MS/MgP 20% MS/MgP
Day 5 1193 1202 1214 1266 1276
Day 10 2294 2362* 2391** 2442** 2451**
Day 20 3417 3543 3635* 3724** 3764**
Notes: Values are expressed as the mean standard deviation. Statistical significance was evaluated in contrast to MgP scaffolds at each time (*P0.05 or **P0.01).
Abbreviations: HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere.

presented in Figure 7, it was obvious that the incorpora- In the SEM morphological analysis (Figure 8), the flake-
tion of MS into MgP scaffold could result in a controllable like apatite crystals were newly produced and distributed on
lysozyme-release pattern without a severe initial burst. The the surface of all MS/MgP composite scaffolds. As with the
slower release of lysozyme took place in the higher HA- quantitative result of calcium ion deposition (Table 1), 20%
containing MS/MgP composite scaffolds; in contrast, the MS/MgP composite scaffolds could generate relatively more
fast release of lysozyme was seen on the bare MgP scaffold, apatite crystals than other MS/MgP scaffolds (Figure 8A).
due to the rapid diffusion. Nevertheless, it was difficult to find morphological variations
Various types of MSs have been incorporated into a between the different HA-containing MS/MgP scaffolds
bulk matrix consisting of either biodegradable polymers (Figure 8B). The elemental analysis of the neoformation
or bioceramics. 20,21 In most cases, the MS-containing of apatite crystals was also carried out by means of EDS.
composite structures were usually aimed to overcome the A representative EDS result is illustrated, in Figure 8A, to
conventional limitations of tissue engineering scaffolds, confirm the noticeable peaks for calcium and phosphate. The
especially poor drug delivery ability. Based on recent pub- EDS patterns were comparable with each other, regardless
lications, it would be feasible to achieve a sustained release of different MS and HA content.
of a single biomolecule or sequential delivery of dual bioac- The effect of HA addition on in vitro calcification of
tive molecules for the tissue engineering application.3,22 In HA-containing MSs was already investigated in our previous
view of clinical applications, antibiotics (eg, gentamicin) study, by soaking them in SBF at physiological conditions.13
or growth factors (eg, bone-morphogenetic protein-2) were We found that the calcium ion deposition was facilitated
encapsulated within poly(lactic-co-glycolic acid) MSs and with increasing incubation time and HA content in MSs.
subsequently mixed with calcium phosphate bone cement This result is consistent with the present work using MS/
to generate a controlled drug delivery system for the effec- MgP composite scaffolds (Tables 1 and 2). The in vitro
tive treatment of bone infections and bone regeneration, calcium deposition was proportional to MS content in MS/
respectively.11,23 MgP scaffold, HA content in MSs, and the incubation time.
Overall, the important role of nanosized HA would be able
In vitro calcification to accelerate the in vitro apatite-forming ability of MS/MgP
The ability of MS/MgP composite scaffolds to initiate in vitro composite scaffolds by SBF treatment. Using morpho-
calcification by SBF treatment was evaluated in two differ- logical and elemental analysis, flake-like apatite crystals
ent aspects: the effect of MS content in MS/MgP scaffolds and noticeable calcium/phosphate peaks were obviously
(Table 1) and of HA content in MS (Table 2). MgP scaffolds detected in all SBF-treated scaffolds, respectively (Figure 8).
contained the lowest calcium ion deposition throughout
Table 2 Quantitative analysis of in vitro calcium deposition was
the entire incubation time. On the other hand, MS/MgP
carried out, in terms of HA content, in 10% MS/MgP scaffolds
composite scaffolds steadily enhanced the in vitro calcium
Ca deposition MS (0% HA)/ MS (38% HA)/ MS (55% HA)/
deposition, while increasing MS content from 2% to 20%.
(g/mL) MgP MgP MgP
At day 20, the 10% and 20% MS/MgP composite scaffolds
Day 5 1242 1231 1266
resulted in the highest quantity of calcium ion deposition Day 10 2344 2401 2442*
(P0.01) (Table 1). With respect to HA content in MS, Day 20 3504 3552 3724**
55% HA-containing MS/MgP composite scaffolds led to the Notes: Values are expressed as the mean standard deviation. Statistical significance
was evaluated in contrast to MS (0% HA)/MgP scaffolds at each time (*P0.05 or
highest level of calcium ion deposition (P0.05), followed **P0.01).
by 38% HA-containing MS/MgP scaffolds (Table 2). Abbreviations: HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere.

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Dovepress Microsphere/magnesium phosphate composite scaffolds

MgP 2% MS (55% HA)/MgP 5% MS (55% HA)/MgP

x1,000 50 m

10% MS (55% HA)/MgP 20% MS (55% HA)/MgP

O P
Mg
Ca
C

0 1 2 3 4
keV

B 10% MS (0% HA)/MgP 10% MS (38% HA)/MgP 10% MS (55% HA)/MgP

Figure 8 SEM measurement of MS/MgP scaffold after SBF treatment for 20 days (1,000 magnification).
Notes: Newly-formed apatite crystals were observed regardless of (A) MS content in MS (55% HA)/MgP scaffolds and (B) HA content in 10% MS. (A) The EDS result was
similar (right bottom).
Abbreviations: EDS, energy dispersive spectroscopy; HA, hydroxyapatite; MgP, magnesium phosphate; MS, microsphere; SBF, simulated body fluid; SEM, scanning electron
microscope.

However, there was no evidence of apatite formation in bare achieve sustained drug-release capacity, biodegradability,
MgP scaffold kept in DI water for the same period of time and bioactivity for effective bone regeneration.
(Figure S1). To date, SBF solution has been widely utilized
for the functionalization of biomaterials as it could mimic the Acknowledgments
actual physiological conditions and consequently, improve This work was supported by the Mid-Career Researcher
both in vitro bioactivity and in vivo performance. To this Program, through a National Research Foundation of Korea
end, many types of biomaterials would be able to create the (NRF) grant funded by the Korea Ministry of Education,
bone-like apatite layers on the surface and integrate with Science, and Technology (MEST) (number 2011-0017572).
surrounding host bony tissues, without forming fibrous
interfacial tissue.24 Disclosure
The authors report no conflicts of interest in this work.
Conclusion
A synergistic combination of MgP scaffold and MS was car- References
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Dovepress Microsphere/magnesium phosphate composite scaffolds

Supplementary material

A B

P
Mg
O

0 1 2 3 4
x1,000 50 m
keV
Figure S1 SEM measurement of the MgP scaffold incubated in DI water for 20 days (1,000 magnification) (A) and the corresponding EDS result (B) were presented as a
negative control for SBF treatment.
Note: No apatite crystals or noticeable Ca peak were detected.
Abbreviations: DI, deionized; EDS, energy dispersive spectroscopy; MgP, magnesium phosphate; SBF, simulated body fluids; SEM, scanning electron microscope.

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