International Journal of Biological Macromolecules 127 (2019) 340–348

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Covalently polysaccharide-based alginate/chitosan hydrogel embedded

alginate microspheres for BSA encapsulation and soft tissue engineering
Lian Xing a,1, Jinchen Sun a,1, Huaping Tan a,⁎, Guoliang Yuan a, Jianliang Li a, Yang Jia a, Dangsheng Xiong a,
Guang Chen a, Jianzhong Lai a, Zhonghua Ling b, Yong Chen b,⁎, Xiaohong Niu c
a
School of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094, China
b
Department of Orthopaedics, Jinling Hospital, Nanjing 210002, China
c
Department of Luoli, Nanjing Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing 210014, China

a r t i c l e i n f o a b s t r a c t

Article history: Hydrogels based scaffolds are very promising materials for a wide range of medical applications including tissue
Received 21 August 2018 engineering and drug delivery. This study reports a covalently cross-linked composite hydrogel embedded with
Received in revised form 8 January 2019 microspheres basing natural polysaccharides as a protein delivery system for soft tissue engineering. This biode-
Accepted 14 January 2019
gradable composite hydrogel derived from water-soluble chitosan and alginate derivatives upon mixing, without
Available online 15 January 2019
addition of chemical cross-linking agents. The gelation is attributed to the Schiff-base reaction between amino
Keywords:
and aldehyde groups of N-succinyl chitosan (N-Chi) and oxidized alginate (OAlg), respectively. Meanwhile,
Hydrogel gel-like microspheres were prepared with a diameter of 2–10 μm by conjugating sodium alginate with Ca2+ in
Microsphere an aqueous emulsion via the emulsion cross-linking technique. Bull Serum Albumin (BSA) was encapsulated
Polysaccharide into alginate gel microspheres and subsequently incorporated into OAlg/N-Chi hydrogels to produce a composite
Tissue engineering scaffold. In the current work, gelation rate, morphology, mechanical properties, swelling ratio, in vitro degrada-
Drug delivery tion and BSA release of the composite scaffolds were examined. The results show that mechanical and stable
properties of gel scaffolds can be significantly improved by embedding alginate microspheres. The alginate mi-
crospheres can serve as a filler to toughen the soft OAlg/N-Chi hydrogels. Compressive modulus of composite
gel scaffolds containing 0.5 mL volume of microspheres was 57.3 KPa, which was higher than the control hydro-
gel without microspheres. Moreover, the controlled release of BSA encapsulated within this composite hydrogels
showed significantly lower rate when compared with control hydrogel or microspheres alone. These character-
istics provide a potential opportunity to use this injectable composite gel scaffold in protein delivery and soft tis-
sue engineering applications.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction In tissue engineering, cell growth factors are water-soluble proteins

and have good effects of promoting the differentiation of stem cells.
Hydrogels are three-dimensionally cross-linked networks, which There is a growing interest in cell growth factor delivery systems that
compose of hydrophilic polymers with high water content [1–3]. Inject- can provide site-specific and controlled release for extended periods
able hydrogels have been used as biodegradable and biocompatible of time. However, cell growth factors always are readily to be denatured
scaffolds to deliver stem cells and protein drugs [4–8]. Polysaccharides in solid microspheres and show burst release from hydrophilic
are very important due to the set of properties not found in other hydrogels, which limit their abilities of inducing stem cells to differenti-
class of biomaterials, which are structurally similar to glycosaminogly- ation. An effective delivery system for controlled release of cell growth
can (GAG) and its analogs [9,10]. Polysaccharide-based hydrogel is a factors is keys to the success of tissue repair. Although some synthetic
kind of high biopolymer with low cross-linking degree, which can ab- microspheres, e.g., PLA and PLGA, have been served as delivery systems,
sorb and maintain a large amount of water. Recently, many polysaccha- proteins would be denatured and lost their bioactivities under the ex-
rides have been employed for the preparation of injectable in situ treme preparation conditions using organic solvents [16,17].
forming hydrogels, including alginate, chitosan, hyaluronic acid and cel- We hypothesize that protein drug that is encapsulated in aqueous
lulose [11–15]. and physiological environment can be more efficiently transported to
a localized site and be released in a sustained-dosage form. Therefore,
⁎ Corresponding authors.
by incorporating with gel-like microspheres, transfer of protein in the
E-mail addresses: [email protected] (H. Tan), [email protected] (Y. Chen). physiological environment can be a valuable prophylactic option. Com-
1
Lian Xing and Jinchen Sun contributed equally to this work. posite hydrogels containing cell growth factors via hydrophilic gel-like

https://doi.org/10.1016/j.ijbiomac.2019.01.065
0141-8130/© 2019 Elsevier B.V. All rights reserved.
 L. Xing et al. / International Journal of Biological Macromolecules 127 (2019) 340–348 341

microspheres can create a more biomimetic microenvironment with the reaction product and the solution was further purified by dialysis
improved biocompatibilities for tissue regeneration. To the best of our (MWCO 14,000) against ultrapure water for 3 days. The purified solu-
knowledge, the synchronous use of hydrophilic gel microspheres with tion was lyophilized at −50 °C and collected as white foam. Percentage
injectable hydrogel basing natural polysaccharides via the Schiff-base oxidation of OAlg was quantified by measuring the number of aldehyde
as cell scaffold for water-soluble drug delivery and soft tissue engineer- using t-butyl carbazate assay.
ing has received limited attention. Herein, we would develop a cova- N-succinyl chitosan (N-Chi) was synthesized according to a reported
lently cross-linked composite hydrogel embedded gel microspheres procedure slightly modified [38]. Chitosan (1.0 g) was added in 57 mL
basing natural polysaccharides, e.g., alginate and chitosan, as a protein deionized water with 3 mL lactic acid. The mixture was stirred for 2 h
delivery system for soft tissue engineering. until chitosan dissolved completely. 240 mL of methyl alcohol and
Alginate is a linear and naturally occurring anionic and hydrophilic 3.0 g of succinic anhydride were added into the mixture solution.
polysaccharide containing blocks of (1–4)-linked β-D-mannuronic acid After stirring 24 h at room temperature, the pH of the solution was ad-
(M) and α-L-guluronic acid (G) monomers [14]. Alginate has been ex- justed to ~7 using 5% (w/v) NaOH solution. The precipitate product was
tensively investigated and used for many biomedical applications due re-dissolved in H2O and purified by dialysis using dialysis tubing
to its biocompatibility, biodegradability and relatively low cost, as well (MWCO 14,000) for 3 days. The final product was freeze-dried at −50
as excellent gel-forming properties by addition of divalent cations °C and collected as white foam.
such as Ca2+ [15–20]. Furthermore, the carbon–carbon bonds of the
cis-diol groups in alginate are able be cleaved and generated reactive al- 2.3. Preparation of gel microspheres
dehyde functions, which can react with amino groups via the Schiff-
base linkage [21–25]. Alginate-based gel microspheres were prepared according to a re-
Chitosan is a linear polymer, partially deacetylated derivative from ported procedure slightly modified [39]. Alginate was dissolved in BSA
chitin. Chitosan has been widely used in drug delivery and tissue engi- to achieve a concentration of 1% (w/v) with a final BSA concentration
neering because of its excellent biocompatibility, biodegradability and of 1% (w/v). The mixture was then added to soybean oil dropwise
antibacterial properties [26–28]. For its strong inter-molecular hydro- while rotate speed at 800 rpm with 4% (w/v) Span 80 as an emulgator.
gen bonding, chitosan has poor solubility in physiological solvents, The emulsion was mixed for 10 min and added 3% (w/v) Tween 80.
which limited its application in tissue engineering. Chemically modified After 5 min, the emulsion was followed by dropwise addition of CaCl2
chitosan structures resulting in improved solubility in physiological sol- solution with a concentration of 8% (w/v). After a further 10 min of
vents have been broadly reported [29–31]. For example, N-succinyl chi- cross-linking, the emulsion was centrifuged at 3000 rpm for 5 min to re-
tosan (N-Chi) is a water-soluble chitosan derivative at various pHs, move the supernatant. The microspheres were washed by isopropanol,
which can be obtained by introduction of succinyl groups into chitosan alcohol and deionized water at room temperature.
N-terminal of the glucosamine units. Recently, N-Chi is attractive as
drug and cell carriers showing biocompatibility and long-term retention 2.4. Hydrogel preparation
in vivo [32–36].
In this study, a polysaccharide-based hydrogel is designed and pre- OAlg/N-Chi hydrogel was cross-linked via the Schiff-base reaction as
pared by the Schiff-base linkages between oxidized alginate (OAlg) a function of OAlg concentration. Dried OAlg was dissolved in 10 mL of
consisting of aldehyde (\\CH_O) and N-Chi consisting of imine (- phosphate buffer solution (PBS, pH = 7.4) to form uniform solutions.
NH2) groups. Alginate gel microspheres loading model protein drug, Concentrations of OAlg solution were fixed as 3%, 5%, 10% and 16% (w/
the Bull Serum Albumin (BSA), were prepared by conjugating alginate v), respectively. 0.25 g N-Chi was dissolved in 10 mL of PBS to form a
with Ca2+ in an aqueous emulsion via the emulsion cross-linking tech- 2.5% (w/v) solution. OAlg and N-Chi solution were completely mixed
nique. Alginate gel microspheres containing BSA were blended with the with the volume ratio of 1:2 at 37 °C to form hydrogels. For incorpora-
OAlg/N-Chi hydrogel to advance biological properties of the composite tion of microspheres, gel microspheres were added to the OAlg solution.
scaffolds. The effects of gel microspheres on characteristics of hydrogels, The microspheres/OAlg mixture was cross-linked with N-Chi solution to
including morphology, gelation, mechanical property, swelling, form hydrogel/microsphere composites at 37 °C. Composite hydrogels
degradation behavior and drug release, were investigated in vitro, with different volume of microspheres were investigated by mechanical
respectively. properties and swelling behavior.

2. Materials and methods 2.5. Characterization of hydrogels

2.1. Materials Swelling capacity of the hydrogels and hydrogel/microsphere com-

posites was performed in PBS at 37 °C. At determined time points, the
Sodium alginate (MW 93 kDa) was purchased from Kelong Chemi- swollen samples were taken out and weight (Ws) after surface adsorbed
cal, Chengdu, China. Chitosan (deacetylation degree: 90%, Mη: 4 water was removed by filter paper. The dry hydrogels were determined
× 105) was purchased from Tianjin Kemiou Chemical Reagent Co., by lyophilizing at −50 °C and weighed (Wd). The swelling ratio was de-
China. Succinyl anhydride was purchased from North China Pharmaceu- fined as (Ws − Wd) / Wd.
tical Goodstar Co., Ltd., China. Ethanol, ethylene glycol and sodium Physiological stability of the hydrogels and hydrogel/microsphere
periodate were purchased from Aladdin Industrial Corporation, Shang- composites was detected by the degradation test with respect to weight
hai, China. All chemicals and reagents were used as received. loss. The weight loss of the scaffolds was monitored as a function of in-
cubation time in PBS at 37 °C under shaking (70 rpm). At determined
2.2. Polymer modification time points, the samples were taken out from the PBS and then the liq-
uid on the surface of hydrogels was sucked up gently by using a piece of
Oxidized alginate (OAlg) was synthesized according to a reported filter paper before being weighed. The weight loss ratio was defined as
procedure [37]. Sodium alginate (3.0 g) was dissolved to 150 mL ultra- (W0 − Wt) / W0 × 100%, and the weight percentage of the remaining
pure water under stirring to obtain a solution of 2% (w/v) concentration. sample was defined as Wt / W0 × 100%.
An aqueous solution of sodium periodate (3.0 g, 10 mL) was added Morphologies of the hydrogels and hydrogel/microsphere compos-
dropwise into the alginate solution and stirred for 24 h at room temper- ites were characterized using scanning electron microscopy (SEM).
ature in the dark. Ethylene glycol (2 mL) was added into the solution to The gel microspheres and composite scaffolds were freeze-dried and
stop the oxidation reaction. Sodium chloride (1.0 g) was dissolved into gold-coated using a Cressington 108 Auto (Cressington, Watford UK).
342 L. Xing et al. / International Journal of Biological Macromolecules 127 (2019) 340–348

Fig. 1. Schematic of incorporation of alginate gel-like microspheres in alginate/chitosan hydrogel. The gelation is attributed to the Schiff-base reaction between amino and aldehyde groups
of N-succinyl chitosan (N-Chi) and oxidized alginate (OAlg), respectively. Alginate gel microspheres containing protein drug were prepared by conjugating alginate with Ca2+ in an aque-
ous emulsion via the emulsion cross-linking technique.

The morphologies were viewed using a Hitachi SU8010 SEM (Hitachi, 2.6. In vitro release study
Japan) operated at 5.0 kV accelerating.
Compression strength of the hydrogels and hydrogel/microsphere In vitro release of BSA from the drug delivery system was analyzed
composites were obtained from uniaxial compression using a Universal for three groups: microspheres, hydrogels and hydrogel/microsphere
Testing Machine (Instron 5943) at 37 °C. Circular samples (15 mm di- composites. Release studies were determined by placing 1 mL samples
ameter, 8.5 mm height) were prepared for compressive tests by in a centrifugal tube containing 10 mL PBS. All samples were maintained
1 mm/min of compression speed. at 37 °C until they were completely degraded or released. The release of

Fig. 2. Pictures to show general morphologies of OAlg/N-Chi hydrogels. (a) Hydrogels with various OAlg concentrations. (b) Hydrogel with 16% OAlg. (c) Hydrogel with 3% OAlg.
 L. Xing et al. / International Journal of Biological Macromolecules 127 (2019) 340–348 343

samples was determined at 280 nm using a Uv/visible spectrophotom-

eter (BioMate 3S, Thermo Fisher Scientific).

2.7. Statistical analysis

Quantitative data were analyzed by software of the Origin 8.0 as

mean values (±standard deviation, SD). Mean values were compared
by use of the independent samples t-test. P values of less than 0.05
were considered to be statistically significant.

3. Results and discussion

3.1. Hydrogel characterization

Alginate gel microspheres containing BSA were prepared by conju-

gating alginate with Ca2+ in an aqueous emulsion via the emulsion
cross-linking technique. Gel microspheres were blended with the
Fig. 3. Swelling of OAlg/N-Chi hydrogels formed by different concentrations of OAlg in PBS
at 37 °C. Values reported are an average of n = 3, ±standard deviation.
OAlg/N-Chi hydrogel to advance biological properties of the composite
scaffolds (Fig. 1). The hydrogels were cross-linked by the Schiff-base
linkages between aldehyde groups (\\CH_O) of OAlg and imine groups
BSA was periodically monitored by extracting 1 mL aliquot and (\\NH2) of N-Chi. The chemical structure of OAlg and N-Chi was also
replenishing with 1 mL PBS. The concentration of BSA in the extracted shown in Fig. 1, which displayed good water soluble property at various

Fig. 4. (a) Stress-strain behavior curves of OAlg/N-Chi hydrogels formed by different concentrations of OAlg at 37 °C. (b) Compression strength of OAlg/N-Chi hydrogels formed by different
concentrations of OAlg at 37 °C. Values reported are an average of n = 3, ±standard deviation.

Fig. 5. SEM of hydrogels with 16% (w/v) OAlg before (a) and after 7 (b), 14 (c), 21 (d) days incubation in PBS at 37 °C. (e), (f), (g) and (h) are the high magnification images of (a), (b),
(c) and (d), respectively.
344 L. Xing et al. / International Journal of Biological Macromolecules 127 (2019) 340–348

pHs. In our study, the determined substitution degree of N-Chi and ox- properties of hydrogels. As a result, the hydrogel with high concentra-
idation extent of OAlg were 36.8% and 44.5%, respectively. tion of OAlg showed higher compression strength than the one with
To optimize properties of delivery system, four different concentra- lower OAlg concentration. Our result demonstrated that the hydrogel
tion of OAlg were used to prepare OAlg/N-Chi hydrogels. The formed with 16% (w/v) OAlg showed the best compression strength since its
hydrogels were displayed in Fig. 2a. These images directly showed cross-linking density was higher than others. This kind of exponential
that the hydrogel with 16% (w/v) OAlg showed a stable and well- relationship could explain the special viscoelasticity [42], which was
shaped appearance (Fig. 2b). Nevertheless, the hydrogel with 3% (w/ not destroyed even when the compression deformation was as high as
v) OAlg was too soft to maintain initial shape (Fig. 2c). Our results dem- 50%. However, the concentration should be in a certain range because
onstrated that a higher content of OAlg results in the formation of stable excess cross-linking density may make gel crisp and lead to the decrease
shape in hydrogels, which is likely due to the comparatively sufficient of compressive modulus of hydrogels.
cross-linking between OAlg and N-Chi. The microstructure of freeze-dried hydrogels with 16% (w/v) OAlg
Swelling properties of the hydrogels are crucial for substance ex- was observed before and after incubation using SEM (Fig. 5). According
change when they are used as injectable scaffolds for drug delivery. to cross-sectional SEM photographs, before incubation, gel and CMs/gel
Fig. 3 illustrated the swilling degree of hydrogels in PBS at 37 °C as a displayed continuous and porous structures (Fig. 5a and e), with the
function of incubation time. The result showed that each kind of hydro- pores being the result of ice crystal formation during the freeze-drying
gel with different concentration of OAlg has good swelling capacity. At process. By measuring pore area using SEM software, the porous struc-
the time of 12 h, the swelling degree of the hydrogel with 16% (w/v) ture in hydrogels was connected with the 30–200 μm of pore size distri-
OAlg was up to 31.1, which means this hydrogel contained about bution. During in vitro degradation of the hydrogel, the internal
96.8% water. Basically, the higher water content makes the hydrogel structure was gradually destroyed by PBS incubation. After 7 days deg-
has higher tissue affinity. The amount of OAlg used in the hydrogel syn- radation, with the broken of cross-linkages, the porous structure of
thesis significantly affected the swelling properties of hydrogels. The hydrogels became more loose (Fig. 5b and f). One can find that the
lower the concentration of OAlg solution is, the higher swelling degree pore diameter became larger by the attack of water, and the hole-wall
of hydrogel formed. In general, an increase in the amount of biopolymer was weaker than the original hydrogels. After 14 days incubation, the
results in a decrease in the water content and weight loss of mass degradation was further evident, as shown in Fig. 5c and g. The porous
[40,41]. Therefore, the hydrogel with 5% OAlg has higher capacity to ab- structure of hydrogels became further looser and the diameter of
sorb water and its swelling degree was significantly higher than the one pores was much larger than those results before incubation (Fig. 5a
with 16% OAlg. and e). At day 21, the hydrogels almost lost their porous structure and
The compression strength of the hydrogels also tested as a function most of the cross-linkages were totally broken in PBS (Fig. 5d and h).
of OAlg concentration at 37 °C, as depicted in Fig. 4. The higher concen- Due to the mass released from matrix during incubation, the hydrogels
tration of OAlg solution was resulted to the increase of cross-linking finally became very weak and soft, that even were not able to be
density in the hydrogels, which improved the mechanical and stable weighted for longer-term study.

Fig. 6. SEM of alginate gel microspheres after freeze-drying at low (a) and high (b, c) magnification.
 L. Xing et al. / International Journal of Biological Macromolecules 127 (2019) 340–348 345

Fig. 7. SEM of hydrogel/microsphere on the surface (a) and inside of composites with 0.1 mL (b), 0.5 mL (c) and 0.9 mL (d) microspheres.

3.2. Incorporation of microspheres in hydrogels volume in the range of 0.1–0.9 mL. The internal morphologies were
not affected by the content ratio of microspheres, whereas no significant
The controlled release of protein delivery technology is an important difference was found between them.
way to make cell growth factor maintain biologically active in vivo envi- Swelling properties are of great significance for drug release and
ronment and to achieve effective long-term release. Our alginate micro- substance exchange in tissue engineering. The swelling degree of hy-
spheres were hydrophilic in aqueous environment, which were readily drogel/microsphere composites with different volume of microspheres
to bind water to form gel-like morphologies. The surface images of the were tested in PBS at 37 °C. As shown in Fig. 8, there were no obvious
gel microspheres loading BSA are presented in Fig. 6. According to
SEM images, the folds and creases were observed on the surfaces of
gel microspheres, which is likely related to the collapse of swollen ma-
trix by virtue of the freeze-drying step. As shown in Fig. 6, most of the
gel microspheres could not maintain their spherical structures and
changed into round sheets (Fig. 6b and c). Obviously the surface change
is attributed to the abrupt dehydration, resembling other natural mac-
romolecular hydrogel system structures. It should be noted that the de-
hydration process obviously reduced the mean size of gel microspheres.
As shown in Fig. 6, the diameter of BSA-loaded gel microspheres was
found in the range of 2–10 μm.
Different volume of alginate gel microspheres were further added to
the hydrogels to form composite scaffolds. Fig. 7 depicted the SEM of the
gel microspheres embedded in the hydrogels with different concentra-
tion of microspheres. We can find that gel microspheres were uniformly
distributed in both surface and internal hydrogels, which were indi-
cated by the arrows. The surface image of the hydrogel with 0.1 mL mi-
crospheres was presented in Fig. 7a. A thin polymer layer can be
observed, which is likely related to the phase segregation of macromol-
ecules by the freeze-drying process, including polysaccharides and BSA,
resulting in the collapse of surface pores. According to cross-section
Fig. 8. Swelling of hydrogel/microsphere composites with different volume of
SEM images (Fig. 7b–d), the hydrogels displayed a continuous and po- microspheres in PBS at 37 °C. Values reported are an average of n = 3, ±standard
rous structure by virtue of the freeze-drying step, with microspheres deviation.
346 L. Xing et al. / International Journal of Biological Macromolecules 127 (2019) 340–348

Fig. 9. (a) Stress-strain behavior curves of hydrogel/microsphere composites with different volume of microspheres in PBS at 37 °C. (b) Compression strength of hydrogel/microsphere
composites with different volume of microspheres in PBS at 37 °C. Values reported are an average of n = 3, ±standard deviation.

differences between the swelling degrees of hydrogel/microsphere The composite hydrogels embedded with different volume of gel
composites with 0.1–0.9 mL volume of gel microspheres. Both hydrogel microspheres were performed compression strength at 37 °C. As
and alginate microspheres have an abundant number of hydrophilic shown in Fig. 9, compression strength of the composites was firstly en-
groups, such as hydroxyl, carboxyl and amino groups, contributing to hanced and then reduced with increasing amounts of gel microspheres.
the water absorption. After incorporation of the microspheres, the com- When the volume of microspheres was fixed as 0.1 mL or 0.3 mL, the
posite hydrogels showed slightly less swelling capacity than the pure compression strength showed as slightly larger than those control pris-
hydrogels. In this study, all the swelling degrees of composite hydrogels tine hydrogels. Our results demonstrated that the composites with
were higher than 25, which meant that all of them contained more than 0.5 mL microspheres have the highest compression strength
96% water. The hydrophilic biopolymer with three-dimensional net- (57.3 KPa) comparing to those samples. As for the 0.5 mL volume of
work structure becomes swelling when it interacts with water. How- OAlg, we speculate that the microspheres filled in the matrix of
ever, the swelling behavior of hydrogels is limited by its cross-linked hydrogels played a reinforcing role, which increased the compression
structure, which depends on the cross-linking density [43,44]. strength of hydrogel/microsphere composites. However, when the

Fig. 10. Images to show various hydrogel/microsphere composites (a) with 0.1 mL (b), 0.3 mL (c), 0.5 mL (d), 0.7 mL (e) and 0.9 mL (f) microspheres before and after compression at 37 °C.
 L. Xing et al. / International Journal of Biological Macromolecules 127 (2019) 340–348 347

Fig. 11. Weight loss of hydrogel/microsphere composites with 0.5 mL volume of Fig. 12. Cumulative release of BSA from hydrogel/microsphere composites with 0.5 mL
microspheres in PBS at 37 °C. Values reported are an average of n = 3, ±standard volume of microspheres in PBS at 37 °C. Values reported are an average of n = 3, ±
deviation. standard deviation.

addition of gel microspheres was higher than the 0.5 mL, the hydrogel/ gel microspheres in hydrogels delayed the burst effect commonly seen
microsphere system would be significantly diluted to form a weak and in the release of BSA from pristine gel microspheres or hydrogels
loose network. alone. The percent cumulative release of BSA in hydrogel/microsphere
The gel microspheres might also tend to move in the compression composites was exceeded ~80% at day 10, which was further increase
process, which would affect the compressive strength of hydrogel/mi- to 92.07 ± 7.04% at day 17. The evaluation of these properties is contrib-
crosphere composites. As seen in Fig. 10, the elastoplasticity of the hy- uted to a further understanding of the formation mechanism of gel scaf-
drogel/microsphere composites had been improved by increasing folds and biological applicability.
volume of alginate gel microspheres, e.g., 0.1–0.9 mL. Comparing to
the 0.1–0.3 mL microspheres (Fig. 10b–e), the hydrogel/microsphere 4. Conclusion
composites with 0.5–0.9 mL gel microspheres have the better compres-
sion strength and excellent elastoplasticity (Fig. 10f–g). The degradation In summary, biodegradable alginate/chitosan hydrogels containing
of hydrogel/microsphere composites with various volume of gel micro- alginate gel-like microspheres were developed for BSA delivery via the
spheres was depicted in Fig. 11. At first stage, the mass of composites Schiff-base reaction. The hydrophilic gel-like microspheres were homo-
was increased by absorbing water. After 24 h, the mass was decreased geneously distributed into hydrogels and act as reinforcing fillers, which
through the degradation as time goes by. Our results demonstrated significantly improved the mechanical properties and stabilized the net-
that the hydrogel composites with 0.5 mL microspheres could be stable work of hydrogels. The controlled release of BSA encapsulated within
until 21 days. The in vitro degradation results were consistent with the composite hydrogels showed significantly lower rate when compared
swelling kinetics and compressive properties of gel scaffolds. These re- with control hydrogel or microspheres alone. This hydrogel/micro-
sult suggested that incorporating microspheres can greatly enhance sta- sphere composite system shows a great potential for a variety of local-
ble and mechanical properties of composite scaffolds, especially the ized controlled drug delivery applications, especially in protein
hydrogel integrated with 0.5 mL microspheres. The embedded micro- delivery. We believe that such a controlled release of delivery system
spheres in hydrogel reduced the water absorbance and improved the for cell growth factors will have a great potential in soft tissue
mechanical properties at the same time. As a result, they were beneficial engineering.
to maintain the structure of composite gel scaffolds and decreased the
degradation rate.
Acknowledgements
3.3. In vitro drug release
We thankfully acknowledge the Jiangsu Province Science and Tech-
nology Support Program of China (BE2013712), the Research Fund for
The in vitro release of BSA from the alginate gel microspheres, the
hydrogels, and the hydrogel/microsphere composites with 0.5 mL mi- the Department of Orthopedics Clinical Research Center of Jiangsu Prov-
ince (BL2012002), the Science and Technology Planning Project of Tian-
crospheres was analyzed in PBS at 37 °C (Fig. 12). Within initial 24 h,
the gel microspheres demonstrated a highest burst release (77.53 ± jin (15ZXZNGX00210) and the Training Project for Leading Talents of
Jiangsu Province Traditional Chinese Medicine (LJ200921).
10.52%) comparing to others. At day 3, the total release of BSA was al-
most 90%. The majority of the BSA was released by day 10 form gel mi-
crospheres. The burst release was delayed in both of the hydrogels and References
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