Nanostructured Architectures by Assembling Polysaccharide ...yal310/papers/Wang-2015-Advanced...Adv....

FULL P

APER

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 927wileyonlinelibrary.com

Nanostructured Architectures by Assembling Polysaccharide-Coated BSA Nanoparticles for Biomedical Application

Zhenming Wang , Kefeng Wang , Xiong Lu ,* Chen Li , Lu Han , Chaoming Xie , Yaling Liu , Shuxin Qu , and Guanming Zhen

DOI: 10.1002/adhm.201400684

Z. Wang, Prof. X. Lu, C. Li, L. Han, C. Xie, Prof. S. Qu, G. Zhen School of Materials Science and Engineering Southwest Jiaotong University Chengdu 610031 , Sichuan , China E-mail: [email protected] Dr. K. Wang National Engineering Research Center for Biomaterials Sichuan University Chengdu 610064 , Sichuan , China Dr. Y. Liu Department of Mechanical Engineering & Mechanics Bioengineering Program, Lehigh University Bethlehem , PA 18015 , USA

Nanostructured architectures are produced on Ti surfaces by layer-by layer (LbL) self-assembling of polysaccharide-coated BSA nanoparticles (BNPs), which created cellular microenvironments mimicking natural extracellular matrix. The BMP-2 encapsulated BNPs are prepared by a desolvation method, and are further coated by chitosan (CHI) coatings to obtain positively charged NPs (CBNPs). Vancomycin (Van) encapsulated CBNPs are obtained by the same method and subsequently coated by oxidized alginate (OALG) to obtain negatively charged NPs (OCBNPs). The CBNPs and OCBNPs are assembled on Ti surfaces to construct nanostructured coatings via electrostatic and covalent interactions. The nanostructured architectures realize the sustained release of BMP-2 and Van for a long term. Bone marrow stromal cells (BMSCs) culture tests confi rm that the bare nanostructured architectures intrinsically facilitate attachment, proliferation, and differentiation of cells, which is attributed to the nanoscale porous structures that are similar to the size of cellular fi lopodia. In-corporating BMP-2 into the nanostructured architectures signifi cantly enhances osteogenetic differentiation of BMSCs, which reveals the synergistic effects of nanostructures and growth factors on cell activity. The antibacterial tests indi-cate that controlled release of Van has good antibacterial ability against Staphy-lococcus epidermidis , while not affecting the normal biological activity of BMSCs.

cell activity. [ 2 ] Cellular behaviors such as attachment, proliferation, migration, and differentiation are dependent on the nano-structures, and the concentration gradients of growth factors in the ECM. [ 3 ] However, it is a great challenge to design microen-vironment with full features of the ECM architecture, which has both nanostruc-tures and suitable growth factors (GFs).

Effi cient immobilization and sustained release of osteogenic growth factors, espe-cially BMP-2, are crucial for regeneration and development of bone tissue because they provide the signals that dictates the fate of different cell populations and their differentiations. [ 4 ] Various methods have been developed to control delivery of growth factors, such as heparin immo-bilization and microspheres encapsula-tion. [ 5 ] Recently, BSA based-nanoparticles (BNPs) have gained considerable attention for their high binding capacity of multiple drugs. BSA is widely used for drug delivery with many advantages such as non-toxicity, biodegradability, biocompatibility, lack of immunogenicity, and extraordinary drug

binding sites in the albumin molecules. [ 6 ] BNPs can preserve the activity of GFs, and minimize the rapid diffusion rate, making them particularly attractive strategies for GF delivery. [ 7 ]

Nanostructures have been included in dental and orthopedic implants, and have shown to improve biocompatibilty and enhance osseointegration. [ 8 ] However, the nanostructures pro-duced by those physical methods, such as acid etching [ 9 ] and anodic oxidation, [ 10 ] are not able to effectively immobilize and control the release of GFs because of the short biological half-life and rapid diffusion of GFs in body fl uid. [ 11 ] Fortunately, nanoparticles (NPs) can be assembled layer by layer to form the nanostructured fi lms. Park et al. [ 12 ] fabricated NP fi lms that are composed of two sets of DNA-modifi ed gold NPs on a glass sub-strate by a LbL deposition method. Fu et al. [ 13 ] have build up the poly(lactic acid) NPs/poly(ethyleneimine) LbL fi lms, which are capable of sustainably releasing pyrene. Patil et al. [ 14 ] assembled multilayer BNPs and poly-dimethyldiallylammonium (PDDAC) fi lms, which are based on the electrostatic interaction between the carboxylic groups of BNPs and amino groups of PDDAC. They also assembled fi lms of BNPs/poly-(acrylic acid) (PAA), which are assembled by the hydrogen bonding interaction between the carboxylic groups of PAA and BSA. Those previous studies indicate that BMP-2 loaded-biodegradable NPs could be

1. Introduction

Constructing nanostructured cellular microenvironment sim-ilar to extracellular matrix (ECM) is an effective approach to pro-mote cell and tissue response to implant surfaces and therefore, enhance implant integration into host bone. [ 1 ] ECM is mainly composed of nanostructured networks that provide structural support for cell attachment, and signaling proteins to regulate

Adv. Healthcare Mater. 2015, 4, 927–937

www.advhealthmat.dewww.MaterialsViews.com

http://doi.wiley.com/10.1002/adhm.201400684

FULL

PAPER

928 wileyonlinelibrary.com © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

used to produce nanostructures that mimic ECM architectures, and realize controlled release of BMP-2 simultaneously.

Bacterial infection is also a critical issue for the implanted devices, which might lead to serious complications and even implant failure. [ 15 ] Immobilizing antibiotics on implant surfaces is an effective route to prevent infection. Various polymeric delivery systems, including silica gel microspheres, [ 16 ] poly(lactic-co-gly-colic acid) microspheres [ 17 ] and heparin-dopamine coatings, [ 18 ] have been developed for controlled release of antibiotics such as vancomycin (Van) and gentamicin. Recently, biodegradable NPs have been used to release the encapsulated drugs over a pro-longed time while reducing the drawbacks of both safety concerns and antibiotic resistance. [ 19 ] Encapsulation of antibiotics into bio-

degradable NPs is a promising strategy for enhancing antibacterial activity without affecting the normal biological functions of cells.

In this study, novel nanostructured architectures were fabri-cated on Ti surfaces by layer-by-layer self-assembling of polysac-charide-coated BNPs. These coatings have nanostructures that are able to enhance cell attachment and spreading. They also realize dual release of BMP-2 and Van for enhancing BMSC functions and antibacterial property, respectively. As shown in Figure 1 , BMP-2 encapsulated BNPs were prepared by a desol-vation method, and were further stabilized by chitosan (CHI) coatings to obtain positively charged NPs (CBNPs). Van encap-sulated CBNPs were obtained by the same method and were subsequently coated by OALG to obtain negatively charged NPs



Figure 1. Schematic illustration of bovine serum albumin (BSA)-based nanoparticles (BNPs) assembled on titanium (Ti) surfaces. a) Chitosan (CHI) coated bone morphogenetic protein-2 (BMP-2) loaded nanoparticles (NPs) (BMP-2-CBNPs) were prepared by a modifi ed desolvation method. First, the mixed BSA/BMP-2 aqueous solution was desolvated by dropwise addition of ethanol to form BMP-2-loaded BNPs. Subsequently, the BMP-2-loaded BNPs were coated with a CHI layer by adding CHI solution under continuous shaking. b) Vancomycin (Van) loaded NPs were obtained by the same method and were subsequently coated by oxidized alginate (OALG) to obtain negatively charged NPs (Van-OCBNPs). c) Cationic CBNPs and anionic OCBNPs with and without drugs were alternatively deposited on the Ti surfaces by layer-by-layer technique. d) The reaction mechanisms between polydopamine (PDA) and CHI, OALG and CHI in this system. (1) The catechol group in the polydopamine matrix can react with the amino groups on CBNPs by means of Schiff-base reaction and Michael-type addition pathway to anchor CBNPs. (2) Free amino groups on the surface of CBNPs and the aldehyde groups of OCBNPs react each other to form Schiff-base covalent bond.

FULL P

APER

929wileyonlinelibrary.com© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

(OCBNPs). Before NP assembling, an intermediate layer of polydopamine was immobilized on the surface of pristine Ti, and the fi rst layer of CBNPs were anchored by covalent interac-tion between the catechol groups of polydopamine and amino groups on CBNPs. [ 20 ] Then, the positively charged CBNPs and negatively charged OCBNPs were assembled LbL to construct nanostructured coatings for BMP-2 and Van release. The pri-mary driving force of the assembling is the electrostatic interac-tion between the highly positively charged CBNPs and highly negatively charged OCBNPs. Another driving force is covalent bond between the amino groups on the surface of CBNPs and the aldehyde groups of OCBNPs. These two interactions results in smooth growth and good stability of the nanostructured BNP coatings. The BMP-2 and Van release from the coatings were characterized. The biocompatibility and antibacterial properties of the nanostructured architectures were evaluated

2. Results

2.1. Characterization of Polysaccharide-Coated NPs

Polysaccharide-coated and bare BNPs were characterized and their particle size, zeta potential and polydispersity index (PDI) are presented in Table 1 . The mean particle size of bare BNPs is

262 ± 11 nm, which is similar to the previously reported value of 260 ± 20 nm in the literature. [ 21 ] The size increases approxi-mately 15 nm per layer after coated by each layer of CHI and OALG. After coated with CHI, the negative zeta potential of the BNPs (–26 mV) changes to positive zeta potential of +30 mV. After coated with OALG, the zeta-potential changes to –35 mV. The uniformity of polysaccharide coatings is indicated by the consistent low PDI after each coating step. In summary, the characteristics of NPs indicate that the CHI and OALG is suc-cessfully coated on the surfaces of the BNPs.

The stability of polysaccharide-coated BNPs is better than bare BNPs, as demonstrated by the PBS immersion test ( Figure 2 ). As shown in Figure 2 a, after 7 days of immersion, the suspen-sion of bare BNPs turns to be clear, which suggests that the bare BNPs are extremely degradable and completely dissolved in PBS. In contrast, the suspension of CBNPs and OCBNPs are milk-white and light yellow, respectively, which indicates that they are still well preserved in the PBS. SEM micrographs reveal that the bare BNPs are swollen and fused with an average diameter of about 1 µm after 1 d in PBS (Figure 2 c), which is larger than their initial size. However, polysaccharides-coated BNPs are spherical in shape, with an average diameter of approximately 260 nm (Figure 2 d,e). After 2-week incubation in PBS buffer and cell culture medium (α-MEM supplemented with 10% FBS and 1% penicillin–streptomycin solution), the diameters of chitosan-coated bovine serum albumin nano-particles (CBNPs) and oxidized alginate-coated bovine serum albumin nanoparticles (OCBNPs) are around 300 nm (Figure S1, Supporting Information). The polysaccharide-coated BNPs are still spherical in shapes. However, the surfaces of the BNPs become fused and rough. These results demonstrate that polysaccharides-coated BNPs are relative stable both in PBS and cell culture medium, which assures stable release of drugs in a long-term study.

The BMP-2 and Van encapsulation effi ciency in polysaccha-rides-coated BNPs are 93.6% and 87.3%, respectively. The high



Table 1. Characteristics of nanoparticles.

Sample Particle size [nm] Polydispersity Zeta potential [mV]

BNPs 262 ± 11 0.054 ± 0.019 –26.77 ± 1.83

CBNPs 277 ± 13 0.056 ± 0.026 30.26 ± 1.26

OCBNPs 289 ± 23 0.058 ± 0.014 –35.70 ± 0.80

BMP-2-CBNPs 272 ± 14 0.062 ± 0.011 31.37 ± 0.33

Van-OCBNPs 287 ± 28 0.071 ± 0.019 –34.62 ± 3.12

Figure 2. Characterization of BNPs and polysaccharide-coated BNPs. a) The photograph of BNPs and polysaccharide-coated BNPs suspension after 7-day incubation in PBS. b) BMP-2 and Van release from CBNPs and OCBNPs. SEM images of c) BNPs, d) CBNPs, and e) OCBNPs after 1-day incu-bation in PBS.

FULL

PAPER


drug encapsulation effi ciency is attributed to the unique molec-ular structure of BSA. BSA has high content of charged amino acids (e.g., lysine, arginine, aspartate and glutamate) in the pri-mary structure and therefore is negatively charged at neutral pH. Thus, BSA could allow the electrostatic adsorption of posi-tive charged BMP-2 or Van without addition of other assistant compounds.

The release profi les reveals that BMP-2 and Van have sus-tained released up to 30 days from polysaccharide-coated BNPs (Figure 2 b). 17.8% ± 3.2% of total BMP-2 are released in the fi rst day, and 81.2 ± 2.5% in the latter period. 18.7% ± 2.4% of total VAN are released in the fi rst day, and 87.3 ± 2.4% in the latter period. The release profi les are fi tted by typical drug release models to elaborate the mechanism for BMP and Van release. There are a series of widely used models, such as the zero order, fi rst order, and Higuchi model. In this study, a simple Peppas model (Equation ( 1) ) is selected to fi t the release profi les:

Q ktn= (1)

where Q is the fraction of total release, k is the kinetic constant, and n is the release exponent. In the spherical sample model, if n < 0.43, diffusion is Fickian; if 0.43 < n < 1, diffusion is non-Fickian; and if n = 1, zero order drug release mechanism domi-nates. The values of n of the BMP and Van release profi les from NPs are shown in Table 2 . The n values of BMP-2 and Van are 0.56 and 0.65, respectively, which reveals that BMP-2 and Van release from NPs are controlled by the non-Fickian transport mechanism.

2.2. Cationic and Anionic BNPs Assembly

The surface features of pristine Ti, alkali-treated-Ti surfaces, PDA-coated Ti surfaces are investigated by SEM. The acid-etched Ti surfaces have micro-pits with sizes in the range of 0.5–5 µm ( Figure 3 a), and alkali treated-Ti surfaces have the nanoporous structures (Figure 3 b). PDA-coated Ti surfaces pre-sent the newly formed PDA nanolayers (Figure 3 c).

SEM micrographs indicate that CBNPs and OCBNPs are successfully anchored onto Ti surfaces layer by layer and form nanoporous structures. After the fi rst layer of CHI-BNPs (2 mg mL −1 ) is deposited, only a small amount of the CBNPs are sparsely dispersed onto the Ti surfaces, and the single CBNPs can be distinguished (Figure 3 d). After more layers of the CBNPs and OCBNPs are assembled, the NPs tend to aggre-gate and cover the surface (Figure 3 e), and fi nally form the nanostructures (Figure 3 f). Increasing the concentration of the NP solution accelerates the assembling process. When the con-centration is increased to 5 mg mL −1 , the one layer of CBNPs already fully covers the Ti surfaces (Figure 3 g). After more NP



Table 2. BMP-2 and Van release kinetics parameters from NPs.

Sample k n r 2

BMP-2-CBNPs 16.79 ± 0.770 0.56 ± 0.024 0.996

Van-OCBNPs 18.26 ± 0.079 0.65 ± 0.030 0.997

Figure 3. Surface morphologies of a) the acid-etched Ti surfaces, b) NaOH-activated Ti surfaces, and c) PDA-coated Ti surfaces. d–i) Surface fea-tures of LbL-assembled NPs with various concentration and assembly layers: d) 2 mg mL −1 , 1 layer, e) 2 mg mL −1 , 10 layers, f) 2 mg mL −1 , 20 layers, g) 5 mg mL −1 , 1 layer, h) 5 mg mL −1 , 10 layers, i) 5 mg mL −1 , 20 layers.

FULL P

APER


layers are assembled, the porous nanostructures seamlessly cover the whole surfaces and form thick fi lms (Figures 3 h,i).

CBNPs and OCBNPs are labeled with fl uorescent dyes and visualized by CLSM to investigate the NP distribution in the coatings, the green FITC- labeled CBNPs (Figure S1a, Supporting Information) and red Rho-labeled OCBNPs (Figure S1b, Supporting Information) are dispersed on the sur-faces, and mixed evenly in the coatings (Figure S1c, Supporting Information). The cross-sectional views reveal that CBNPs and OCBNPs are homogeneously mixed in the middle of the coat-ings (Figures S1e–g, Supporting Information). The thickness of the 20-layer fi lms is about 10 µm.

2.3. Dual Drug Release from Nanostructured Architectures

The release profi les of BMP-2 and Van from nanostructured architectures exhibit sustained release up to 6 weeks. The BMP-2 release from Ti/BNP-BMP-2&Van and Ti/BNP-BMP show similar behaviors ( Figure 4 a). BMP-2 is quickly released in the fi rst 6 days, followed by sustained release in the latter

period. During the fi rst 6 days. 34.29 ± 3.2% and 32.8 ± 2.6% of BMP-2 were released from Ti/BNP-BMP-2&Van and Ti/BNP-BMP-2 substrates, respectively. The release profi les of BMP-2 were also fi tted by the Peppas model. In the thin fi lm model, if n < 0.5, diffusion is Fickian; if 0.5 < n < 1, diffusion is non-Fickian; and if n = 1, zero order drug release mechanism domi-nates. The values of n are 0.49 and 0.48 ( Table 3 ), respectively, which indicates that BMP release is dominated by a Fickian dif-fusion mechanism.

The Van release from Ti/BNP-BMP-2&Van and Ti/BNP-Van shows almost the same behavior (Figure 4 b). Both of them shows stable release rate during the whole period. After 6 weeks, the Van from Ti/BNP-BMP-2&Van and Ti/BNP-Van are 76.8 ± 1.9% and 78.7 ± 1.2%, respectively. The values of n are 0.58 and 0.59 ( Table 4 ), respectively, which indicates that Van release is dominated by a non-Fickian diffusion mechanism. The comparison of BMP-2 from Ti/BNP-BMP-2&Van and Ti/BNP-BMP-2, and Van from Ti/BNP-BMP-2& Van and Ti/BNP-Van, indicates that the growth factors and antibiotics do not interfere each other, and can be released from the nanostruc-tured coatings independently. These results suggest that the current nanostructured coatings are suitable to delivery mul-tiple types of drugs and preserve their own characteristics.

2.4. Antibacterial Tests

Antibacterial activity of Pristine Ti, Ti/BNP, Ti/BNP-BMP-2, Ti/BNP-Van, and Ti/BNP-BMP-2&Van was evaluated against S. epidermidis by comparing the inhibition of bacterial cells. As shown in Figure 5 a, bacteria cells on pristine Ti, Ti/BNP and Ti/BNP-BMP-2 proliferate during the 7-day culture and sus-pensions are turbid, indicating that they have no antibacterial activity. The bacteria cells on Ti/BNP-Van and Ti/BNP-BMP-2&Van are effectively and signifi cantly inhibited and the sus-pensions are clear, which proves that Van is released and works against bacteria. The quantitative tests indicate that the bacte-ricidal ratios of Ti/BNP-Van and Ti/BNP-BMP-2&Van are 96% and 95%, respectively (Figure 5 b).

2.5. In Vitro Cell Culture

The morphology and attachment of the BMSCs on nanostruc-tured coatings after 3-day culture are imaged by SEM ( Figure 6 ).



Figure 4. a) BMP-2 and b) Van release from various nanostructured architectures on Ti surfaces.

Table 3. BMP-2 release kinetics parameters obtained from the coatings.

Sample k n r 2

Ti/BNP-BMP-2&Van 11.56 ± 1.88 0.49 ± 0.057 0.953

Ti/BNP-BMP-2 12.25 ± 1.83 0.48 ± 0.052 0.972

Table 4. Van release kinetics parameters obtained from the coatings.

Sample k n r 2

Ti/BNP-BMP-2&Van 10.08 ± 0.55 0.58 ± 0.02 0.997

Ti/BNP-Van 8.27 ± 0.071 0.59 ± 0.03 0.994

FULL

PAPER


The results reveal that the density of BMSCs on the nanostruc-tured surfaces is higher than that on pristine Ti surfaces. The BMSCs spread better on those nanoporous surfaces, with more stress fi bers and fi lopodia attachments (Figures 6 c,e,g,i). High magnifi cation images show that BMSCs on nanostructured sur-faces have a close contact with the BNPs and present long thin pseudopodia, which are similar to the size of NP and interact with NPs (Figures 6 d,f,h,j). On the other hand, BMSCs on pris-tine Ti surfaces still display a stellate shape with fewer stress fi bres and weaker adhesions (Figure 6 b).

The results of Alamar blue tests indicate that BMSCs pro-liferation on all nanostructured coatings (with and without BMP-2) was signifi cantly greater than the pristine Ti surfaces ( Figure 7 ). The result implies that the BNP assembled nano-structures signifi cantly promote the proliferation of the BMSCs. Cell proliferation on Ti/BNP-Van and Ti/BNP-BMP-2&Van sur-faces is not signifi cantly different to that on Ti/BNP and Ti/BNP-BMP surfaces, which indicate that the addition of Van in the nanostructured coatings does not hinder cell proliferation.

ALP is an early-stage marker of BMSCs differentiation to osteoblasts. As shown in Figure 8 , the ALP activity of BMSCs on BNP-assembled Ti surfaces (Ti/BNP) is higher than that on pristine Ti, which indicates the BNPs assembled

nanostructures are benefi cial for the differentiation of the BMSC. The ALP activity of BMSCs on BMP-2 incorporating coatings (Ti/BNP-BMP-2, Ti/BNP-BMP-2&Van) is signifi cantly higher than that on the counterparts without BMP-2 (Ti/BNP, Ti/BNP-Van), which indicates that the BMP-2 encapsulated coatings provide a favorable environment for osteogenetic dif-ferentiation of BMSCs. There was no signifi cant difference in ALP activity level on the coatings with (Ti/BNP-Van, Ti/BNP-BMP-2&Van) and without Van (Ti/BNP, Ti/BNP-BMP-2), which reveals that the differentiation of BMSCs is not affected by the addition of Van.

3. Discussion

Nanostructured architectures were produced on Ti surfaces by LbL assembly of CBNPs and OBNPs, which creates cel-lular microenvironments mimicking ECM. These bio-insipired nanoporous coatings have a few advantages: (1) the nanostruc-tured features on substrates enhances the attachment and dif-ferentiation of BMSCs. (2) The nanostructured coatings realize the sustained dual release of BMP-2 and Van. (3) The syner-gistic effect of nanostructures and release of BMP-2 on cell behaviors are achieved. (4) The introduction of antibiotics into nanostructured coatings enhances antibacterial activity without affecting normal biological functions of BMSCs.

The success of the nanostructured architectures are highly dependent on the unique characteristics of polysaccharides coatings on BNPs. LbL on NPs is a versatile platform for tuning tissue specifi city and therapeutic effi cacy, in a controlled manner. [ 22 ] In this study, BNPs are stabilized by the CHI coat-ings, which endow the BNPs with a stable structure and sus-tained release of proteins and drugs. Generally, BNPs are sta-bilized by glutaraldehyde or genipin, which are cytotoxic and has the possibility to react undesirably with the therapeutic agents entrapped within the NP. Therefore, coating BNPs with polymers, instead of utilizing hazardous crosslinkers, is a supe-rior way to stabilize the BNPs and protect them against enzy-matic degradation. Various cationic polymer coatings have been used to coat on negative charged BNP, such as poly- L -lysine, [ 21 ] polyethylenimine (PEI) [ 7a ] and poly(allylamine hydrochloride) (PAH)/sodium poly(4-styrene sulfonate) (PSS). [ 23 ] In this study, CHI is used to coat on BNP surfaces, which is a polycationic polymer with excellent biocompatibility and admirable biodeg-radability. [ 24 ] The primary amines in the CHI backbone can bind to negatively charged BNPs via an electrostatic interac-tion and lead to the spontaneous formation of nano-thick fi lms on the BNPs surfaces, [ 25 ] which improve the stability of BNPs (Figure 2 ) and realize the sustained release of BMP-2 and Van for a long term (Figure 2 b).

Second, the OALG coatings change the CHI-coated BNPs from positive to negative charge, and also endow the NP sur-faces with enough reactive sites, including aldehyde and car-boxyl groups. Alginate is an anionic polysaccharide, which has recently gained recent interest in the fi eld of wound care and tissue engineering for its superior biocompatibility. [ 26 ] After alginate is oxidized by sodium periodate, the carbon-carbon bonds in the vicinal glycols of alginate are cleaved and change to dialdehyde groups, which can react with the free amino groups of CHI and form Schiff-base bond, as shown in Figure 1 . [ 27 ] In



Figure 5. a) Antibacterial activity of various nanostructured protein fi lms against S. epidermidis after 7 days of incubation. b) Optical density meas-urement of growth of S. epidermidis .

FULL P

APER


addition, there are also strong electrostatic interactions between anionic OALG and cationic CHI-coated CBNPs. Both covalent Schiff-base bonds and electrostatic interactions contribute to the

tight OALG/CHI coatings and result in the sustained release of Van. The covalent Schiff-base bond and electrostatic interactions are also the basis of LbL assembling of CBNPs and OCBNPs (Figure 1 ).

The nanostructured architectures promote the attachment, proliferation and differen-tiation of BMSCs even without BMP incor-poration, which indicates that these nano-structures intrinsically favor cell activity. As revealed by SEM micrographs, cells extend their fi lopodia, which are in similar size as NP, and interact with and attach on the NPs. It should be noted that BSA itself is a non-adhesive protein that does not facilitate cell adhesion. However, the BNP assembled nanostructures not only contain cell adhesive CHI, but also have nanoporous structures that are mimic ECM architectures. The nano-structures increase the roughness and spe-cifi c surface area, which improves cell attach-ment and enhances cell performance. [ 28 ] It is well documented that nanostructures could enhance cell activity. For instance, Klein et al. [ 29 ] revealed that there are syner-gistic effects between submicron structures, which are created by an acid-etched method, and surface hydrophilicity on osteogenetic cell adhesion and maturation. Cai et al. [ 30 ] suggested that the surface nanostructured substrates, which are produced with surface mechanical attrition treatments), are ben-efi cial for the activity of BMSCs, including adhesion, fi lament orientation, prolifera-tion and gene expression. Webstera et al. [ 31 ] confi rmed that the attachments and growth rates of endothelial cells are improved by the sub-micron surface features of Ti implants produced by electron beam evaporation. Liu et al. [ 32 ] studied the infl uence of patterned substrate geometry on cell adhesion and suggested that nanostructures enhances fi lopodia generation and cell adhesion. Our studies further confi rm the benefi ts of adding nanostructures on implant surfaces, which improves the interaction between implant surfaces and cells/tissue.

The nanostructured architectures realize sustained release of BMP-2 and preserve its activity. The release of BMP-2 from the nanostructured coatings exhibited a low ini-tial burst release and a sustained release for 42 days (Figure 5 a) The nanostructures with BMP-2 leads to much higher ALP activity of BMSCs than the bare nanostructures, which reveals that BMP-2 in the coatings preserve its osteogenetic activity with prolonged oste-

oinductivity. Many previous investigations reported that the initial burst release followed by sustained release is benefi cial for promoting new bone formation. For example, Liu et al. [ 33 ]



Figure 6. SEM images of BMSCs on a,b) Ti, c,d) Ti/BNP, e,f) Ti/BNP -Van, g,h) Ti/BNP-BMP-2, and i,j) Ti/BNP-BMP-2&Van for 3 days. The micrograph in the right column is the amplifi ed one of that in the left column.

FULL

PAPER


evaluated BMP-2 release from gelatin sponge loaded BMP-2/2-N, 6-O-sulfated chitosan nanoparticle, and the results indi-cated that the system exhibits an initial burst release followed by a sustain release for 21 days, and the system has higher ALP activity than the system without drug delivery carriers. Guelcher et al. [ 34 ] demonstrated BMP-2 from polyurethane pow-ders show a initial burst release followed by a sustained release for 21 days, which promoted the ALP activity of MC3T3 cells. In summary, our developed nanostructured architectures are capable of programmed delivery of BMP-2, and other growth factors, for bone regeneration.

The results of in vitro tests indicated that the nanostruc-tured coatings with BMP-2 signifi cantly enhance osteoge-netic differentiation of BMSCs, which reveals the synergistic effects of nanostructures and growth factors on cell activity. During cell culture, the bioinspired nanostructures provide extraordinary active sites for cell attachment and prolifera-tion. BMP-2 is sustainably released from the nanostructures to initiate signaling cascades that ultimately regulate BMSC

differentiation to osteoblasts. Cho et al. [ 35 ] indicated that appropriate nanostructural stimulation combined with BMP-2 signals amplifi es osteoinductive signals and enhances human mesenchymal stem cells osteogenetic differentiation. Lai et al. [ 36 ] confi rmed that surface-functionalized TiO 2 nanotubes with BMP-2 immobilization synergistically promotes the dif-ferentiation of mesenchymal stem cells. The current results are consistent with those reports and reveals the importance of the synergistic effects of nanostructures and signaling proteins.

Developing antibacterial materials without compromising biological functions is critical for the clinical success of implant devices. The current results demonstrated that the presence of the Van in the coating endows Ti surfaces with excellent antibacterial ability, which does not interfere with BMSC activities. Van in the nanostructured coatings exhibits low initial burst release and sustained release at latter period. The low initial burst release of Van from the nanostructured coatings at subtoxic concentrations is desirable for the treat-ment of bone infections. Moreover, local sustained delivery of antibiotics from the bone devices for 6–8 weeks is antici-pated to protect the surface from bacterial colonization until the site is vascularized, and reduce the possibility of secondary complications. [ 37 ] Various carrier materials have been used for controlling Van release, including bioactive borate glass, cal-cium sulfate pellets and silica xerogel. [ 38 ] Most of them have been shown to produce burst releases that results in Van levels above the maximum toxic concentration in the initial periods. In the present study, the burst release from Ti/BNP-BMP-2&Van did not exceed 10% of the total Van content of the mate-rial. It also maintains the effect concentrations of Van for more the one month, which would reduce the risk of infection for a long-term. In summary, the result suggests that the encapsu-lation of Van in the coatings could inhibit the long-term pro-liferation of bacterial without sacrifi cing the normal biological functions of BMSCs.

4. Conclusions

Nanostructures architectures were prepared by LbL assembly of polysaccharide-coated BNPs. SEM and CLSM measure-ments demonstrated that BNPs were successfully anchored onto Ti surfaces. The nanostructured coatings realize the sustained release of BMP-2 and Van for a long term. The BMP-2 and Van do not interfere with each other, and can be released from the nanostructured coatings independently. These results suggest that the current nanostructured archi-tectures are suitable to deliver multiple types of drugs and preserve their own characteristics. The results of in vitro tests confi rmed that the nanostructured coatings signifi cantly pro-mote the attachment, spreading, proliferation, and differenti-ation of BMSC. Importantly, the nanostructured coatings with BMP-2 signifi cantly enhance osteogenetic differentiation of BMSC, which reveals the synergistic effects of nanostructures and growth factors on cell activity. The antibacterial tests indi-cated that controlled release of Van has good antibacterial ability against S. epidermidis , while does not affect the normal biological activity of BMSC.



Figure 7. Cell proliferation of BMSCs on the various nanostructured pro-tein fi lms after 3 and 7 days of culture.

Figure 8. ALP activity of BMSCs on the various nanostructured protein fi lms after 7 and 14 days of incubation.

FULL P

APER


5. Experimental Section Materials : Commercial pure Ti (Baoji Special Iron and Steel

Co., Ltd., China) was cut into discs (10 mm in diameter, 1 mm in thickness). Chitosan (low molecular weight, deacetylation degree 98%), alginate, dopamine hydrochloride, bovine serum albumin (BSA), NaIO 4, fl uorescein-5-isothiocyanate (FITC), and rhodamine (Rho) were purchased from Sigma–Aldrich (USA). Recombinant human bone morphogenetic protein-2 (BMP-2) was purchased from Rebone Biological Technology Ltd., Shanghai, China. Enzyme-linked immunosorption assay (ELISA) kits were purchased from R&D Ltd., USA. The BCA assay kits and ALP Assay Kits were purchased from Nan Jing Jian Cheng Ltd., China. Fetal bovine serum (FBS), α-MEM and 1% penicillin−streptomycin solution were purchased from HyClone (USA). Medium 199 is from GIBCO (USA). All other regents and solvents were of reagent grade. Oxidized alginate (OALG) was obtained by the oxidation reaction with sodium periodate (NaIO 4 ).

Preparation of Drug-loaded NPs : Cationic NP preparation: CHI-coated BMP-2 loaded BNPs were prepared by a modifi ed desolvation method as previously described. [ 7b ] As shown in Figure 1 a, the process includes three steps. First, BSA (50 mg) was dissolved in aqueous NaCl solution (5 mL, 10 × 10 –3 M ) under constant stirring (600 rpm) at room temperature for 15 min. BMP-2 solution (3 mL, 0.5 mg mL −1 ) in double distilled H 2 O was added into the above solution and incubated for 4 h. Second, this mixed BSA/BMP-2 aqueous solution was desolvated by dropwise addition of ethanol (20 mL). The mixture was stirred (600 rpm) under room temperature for 12 h and BMP-2 encapsulated BNPs (BMP-BNPs) were formed. Finally, the BMP-2-BNPs were then coated with a CHI layer by addition of CHI solution (25 mL, 0.5 mg mL −1 , pH 5.0) under continuous shaking for 0.5 h. The CHI-coated BMP-2-BNPs were obtained by centrifugation (15 000 rpm for 15 min) and freeze-drying, which were denoted as BMP-2-CBNPs.

Anionic NP Preparation : The preparation processes of OALG-coated Van-CBNPs were shown in Figure 1 b. First,BSA (200 mg) and Van20 (mg) were dissolved in of aqueous NaCl solution (20 mL, 10 × 10 –3 M ). Second, ethanol (80 mL) was added dropwisely to the mixed BSA/Van aqueous solution under stirring (600 rpm) for 12 h. Third, the Van-BNPs were then coated with a CHI layer by addition the CHI solution (100 mL,0.5 mg mL −1 , pH 5.0) under continuous shaking for 0.5 h. The CHI-coated Van-BNPs were purifi ed by centrifugation and freeze-drying, and named as Van-CBNPs. Fourth, the Van-CBNPs were further coated with OALG by re-suspending them in an OALG aqueous solution (0.5 mg mL −1 ) to form a NP suspension (4 mg mL −1 ). After incubated for 15 min, the OALG-coated Van-CBNPs were obtained by centrifugation and freeze-drying. The anionic OALG-coated CBNPs were denoted as OCBNPs. Blank CBNPs and OCBNPs without drug encapsulation were prepared in the same way, which were used as the control groups. To prepare FITC-CBNPs and Rho-OCBNPs, CHI was labeled with FITC or Rho according to the method described in our previous publications. [ 39 ]

Characterization of NPs : The mean particle size, polydispersity index (PDI), and zeta potential was calculated using the Smoluchowski model with a laser particle analyzer (ZETA-AIZER,Malvern,UK). All samples were tested in triplicate, and the mean values and standard deviation were calculated. The morphology of NPs was examined with a scanning electron microscope (SEM; JSM 6390, JEOL, Japan). The samples for SEM observation were prepared by dropping the NP suspension (10 µL) on a silicon wafer and dried at an ambient atmosphere. FITC-CBNPs and Roh-OCBNPs were observed by a confocal laser scanning microscope (CLSM, TCSSP5, Lecia, Germany). The drug encapsulation effi ciency of NPs (%) was determined based on the ratio of the encapsulated amount to the initial amount of drugs (BMP-2 and Van) according to Equation ( 2) : [ 40 ]

= − ×Encapsulation efficiency (%)

Total drug Free drugTotal drug

100

(2)

Ti Substrate Preparation : First, the Ti discs were mechanically polished using a series of SiC papers (240, 400, and 800 grit). Second, the discs were etched in a mixed acid (H 2 SO 4 /HCl/H 2 O = 1:1:1, volume ratio)

at 60 °C for 3 h to remove natural oxide layers and increase surface roughness. Third, the discs were alkali-treated to increase the hydrophilicity by immersing them in 100 mL of 5 M NaOH aqueous solution at 60 °C for 3 h, and then thoroughly rinsed with DI water and blown dry. Finally, the NaOH-activated Ti substrates were coated with a thin adherent polydopamine (PDA) fi lm by immersing them in the dopamine solution (2 mg mL −1 , 2 mL) for up to 18 h. Dopamine solution was prepared by dissolving dopamine in Tris-HCl buffer solution (10 × 10 –3 M , pH 8.5). The PDA fi lms contain catechol, amino, and carboxylic acid groups, which exhibits latent reactivity toward amine so as to anchor the as-prepared NPs.

NPs Assembly on Ti Surfaces : A schematic illustration of BNPs assembly Ti substrates is shown in Figure 1 c. Cationic CBNPs and anionic OCBNPs were alternatively deposited on the Ti discs layer-by-layer. First, the PDA functionalized Ti discs were placed in the cationic BMP-2-CBNP suspensions with different concentrations (2 mg mL −1 , 5 mg mL −1 ) for 15 min and subsequently rinsed with pure water. Second, the as-treated Ti discs were placed in the anionic Van-OCBNP suspensions with different concentrations (2 mg mL −1 , 5 mg mL −1 ) for 15 min, followed by the same rinsing procedures. By repeating the above two steps, multilayers of cationic and anionic NPs were assembled to produce a stable nanostructured coating containing BMP-2 and Van through electrostatic and covalent interaction between each layer. The specimen was named as Ti/BNP-BMP-2&Van. Control cases that contain only BMP-2, Van and bare BNPs were also fabricated in the same way, which are named as Ti/BNP-BMP-2, Ti/BNP-Van, and Ti/BNP respectively. The NPs used for assembling different coatings is listed in Table 5 . The fi nal products were stored at 4 °C for further usage. Each specimen contained 153 ng of BMP-2 and 348 µg of Van, which was calculated according to the total drugs release from the coatings after two months.

Drug Delivery Studies : The BMP-2 and Van release from polysaccharide-coated BSA-NPs were determined to study the effect of polysaccharides coating on the drug release. The BMP-2 or Van encapsulated NPs were incubated in the PBS (2 mL, 2.0 mg mL −1 , pH 7.4) at 37 °C under continuous shaking. At predetermined intervals, the samples were centrifugation and the supernatants were collected for analysis. The BMP-2 and Van release from NP assembled coatings were also evaluated. Each specimen was soaked the PBS (2 mL, pH 7.4). At predetermined time intervals, the release medium was collected and replaced with fresh PBS buffer. The amount of BMP-2 released into the medium was determined by the ELISA kit according to the instructions of the manufacturer. The measurements were done with a microplate reader (MQX200, BioTEK, USA). The concentration of Van released at each time point was determined by measuring the absorbance at 280 nm by an UV–vis spectrophotometer (Lambda 35, Perkin-Elmer, Germany).

Antibacterial Activity Assay : Antibacterial activity assays were carried out using Staphylococcus epidermidis (ATCC 12228), the most common microbial pathogen encountered in orthopedic infections. S. epidermidis was cultivated in Luria-Bertani (LB) broth. First, Pristine Ti, Ti/BNP, Ti/BNP-Van, Ti/BNP-BMP-2 and Ti/BNP-BMP-2&Van were placed in a 24-well plate, and the bacterial suspension (100 µL, 106 CFU mL −1 )



Table 5. Nanostructured architectures prepared by the assembly of dif-ferent NP pairs. a)

Samples Bare CBNPs BMP-2-CBNPs Bare OCBNPs Van-OCBNPs

Ti/BNP √ √

Ti/BNP-BMP-2 √ √

Ti/BNP-Van √ √

Ti/BNP-BMP-2&Van √ √

a) Notes: BNPs, BSA nanoparticles; CBNPs, CHI-coated BNPs; OCBNPs, OALG-coated CBNPs; Bare CBNPs, CBNPs without drug; Bare OCBNPs, OCBNPs without drug.

FULL

PAPER


was added onto the surfaces of each sample. Next, the samples were incubated for 12 h at 37 °C in an incubator. Afterward, the LB broth (900 µL) was added to each well in the plates, and the samples were further incubated for 12 h at 37 °C. Finally, the bacterial suspension (200 µL) on each sample was collected and transferred to a 96-well plate. Bacterial growth was monitored by measuring optical density (OD) at 600 nm in the microplate reader. The bactericidal ratio of the coatings was calculated according to Equation ( 3) : [ 41 ]

×

Bactericidal ratio(%) =OD of contrastivegroup-OD of experimental group

OD of contrastive group100

(3)

BMSC Culture Study : Bone marrow stromal cells (BMSCs) were cultured on the as-prepared coatings to evaluate their cytocompatibility. BMSCs were extracted from 1-week-old Sprague–Dawley rats as described in our previous study. [ 42 ] BMSCs (passage 4) were seeded on the scaffolds at a density of 2 × 10 4 cells/sample and cultured in α-MEM supplemented with 10% FBS and 1% penicillin−streptomycin solution at 37 °C in a 5% CO 2 incubator. For cell attachment study, the cells on the specimens were washed twice with PBS, and then fi xed with 2.5% glutaraldehyde for 4 h at room temperature after 3 and 7 days of incubation. The cells were then subjected to step dehydration with a graded series of ethanol/water solutions (30%,70%,90%, 100%, and 100%) for 10 min each step. Finally, the cells were dried by critical point drying and gold-sputtered prior to SEM observation. Cell proliferation was evaluated by the Alamar Blue assay after 3 and 7 days of culture. First, the culture medium in the 24-well plate was replaced with Alamar blue regents (0.4 mL of Medium 199 without phenol red supplemented with 10% FBS and 10% Alamar Blue). After 4 h incubation, reagents were carefully transferred to 96-well plates. The optical density was measured at 570 and 600 nm against a medium-blank Alamar Blue using the microplate reader. Cell differentiation was evaluated by the alkaline phosphatase activity (ALP) assay. After 7 and 14 days of culture, the medium was removed from the wells. Subsequently, the cells on the scaffolds were washed twice with PBS and lysed with the Triton X-100 (200 µL, 1.0% v/v). The lysate was centrifuged and the supernatant was employed for ALP activity determination. The fi nal ALP activity was normalized with respect to the total protein content obtained from the same cell lysate. The total protein concentration of the cell lysate of each sample was measured using the BCA Kit. ALP activity was measured using the ALP Assay Kit. In each case, four specimens were tested and the assay was repeated three times.

Statistical Analysis : The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey multiple-comparison post hoc test to determine the signifi cance of difference between the test groups. The level of statistical signifi cance was set as p ≤ 0.05.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This project was fi nancially supported by the 863 Program (2015AA031301), Sichuan Youth Science-Technology Foundation (2011JQ0010), Construction Program for Innovative Research Team of University in Sichuan Province (14TD0050), SRTP (201410613001), and education program for Innovation Entrepreneurship of Southwest Jiaotong University. Y.L. acknowledges the support of National Science Foundation (NSF) Grant No. CBET-1113040, CBET-1264808.

Received: November 4, 2014 Revised: January 8, 2015

Published online: February 5, 2015

[1] K. Liu , Y. Tian , L. Jiang . Prog. Mater. Sci. 2013 , 58 , 503 . [2] a) T. Dvir , B. P. Timko , D. S. Kohane , R. Langer , Nat. Nanotechnol.

2011 , 6 , 13 ; b) C. Frantz , K. M. Stewart , V. M. Weaver . J. Cell Sci. 2010 , 123 , 4195 .

[3] A. Shekaran , A. J. García , J. Biomed. Mater. Res., Part A 2011 , 96 , 261

[4] a) D. Ben-David , S. Srouji , K. Shapira-Schweitzer , O. Kossover , E. Ivanir , G. Kuhn , R. Müller, D. Seliktar, E Livne , Biomaterials 2013 , 34 , 2902 ; b) R. Tejero , E. Anitua , G. Orive , Prog. Mater. Sci. 2014 , 39 , 1406 .

[5] a) S. E. Kim , S. H. Song , Y. P. Yun , B. J. Choi , I. K. Kwon , M. S. Bae , H. J. Moon , Y. D. Kwon , Biomaterials 2011 , 32 , 366 ; b) M. Li , X. Liu , X. Liu , B. Ge , Clin. Orthop. Relat. Res. 2010 , 468 , 1978 .

[6] F. Kratz , J. Controlled Release 2008 , 132 , 171 . [7] a) S. Zhang , G. X. Wang , M. Lin , H. P. Chatzinikolaidou ,

M. Jennissen , M. Laub , H. Uludag , Biotechnol. Prog. 2008 , 24 , 945 ; b) S. Zhang , C. Kucharski , M. R. Doschak , W. Sebald , H. Uludag , Biomaterials 2010 , 31 , 952 .

[8] a) M. Domanski , R. Luttge , E. Lamers , X. F. Walboomers , L. Winnubst , J. A. Jansen , J. G. E. Gardeniers , Nanotechnology 2012 , 23 , 065306 ; b) L. Prodanov , E. Lamers , M. Domanski , R. Luttge , J. A. Jansen , X. F. Walboomers . Biomaterials 2013 , 34 , 2920 .

[9] L. Zhao , S. Mei , P. K. Chu , Y. Zhang , Z. Wu , Biomaterials 2010 , 31 , 5072 .

[10] X. Huang , Z. Liu , Surf. Coat. Technol. 2013 , 232 , 224 . [11] Y. Takahashi , M. Yamamoto , Y. Tabata , Biomaterials 2005 , 26 , 4856 . [12] Z. G. Estephan , Z. Qian , D. Lee , J. C. Crocker , S. J. Park , Nano Lett.

2013 , 13 , 4449 . [13] Y. H. Jiao , Y. Li , S. Wang , K. Zhang , Y. G. Jia , Y. Fu , Langmuir 2010 ,

26 , 8270 . [14] V. Mohanta , S. Patil , Langmuir 2013 , 29 , 13123 . [15] Y. Tian , H. Cao , Y. Qiao , F. Meng , X. Liu , Acta Biomater. 2014 , 10 ,

4505. [16] C. G. Ambrose , G. R. Gogola , T. A. Clyburn , A. K. Raymond ,

A. S. Peng , A. G. Mikos , Clin. Orthop. Relat. Res. 2003 , 415 , 279 . [17] S. Radin , T. Chen , P. Ducheyne , Biomaterials 2009 , 30 , 850 . [18] D. W. Lee , Y. P. Yun , K. Park , S. E. Kim , Bone 2012 , 50 , 974 . [19] M. H. Xiong , Y. Bao , X. Z. Yang , Y. H. Zhu , J. Wang , Adv. Drug

Delivery Rev. 2014 , 78 , 63. [20] Y. Liu , K. Ai , L. Lu , Chem. Rev. 2014 , 9 , 5057 . [21] D. Li , X. Lu , H. Lin , F. Ren , Y. Leng , J. Mater. Sci. 2013 , 24 , 489 . [22] M. Rajam , S. Pulavendran , C. Rose , A. B. Mandal , Int. J. Pharm.

2011 , 410 , 145 . [23] C. Xie , X. Lu , K. Wang , F. Meng , O. Jiang , H. Zhang , W. Zhi , L. Fang ,

ACS Appl. Mater. Interfaces 2014 . 11 , 8580 . [24] L. Han , H. Lin , X. Lu , W. Zhi , K. f. Wang , F. Z. Meng , O. Jiang ,

Mater. Sci. Eng. C. 2014 , 40 , 1 . [25] H. D. Singh , G. Wang , H. Uludag , L. D. Unsworth , Acta Biomater.

2010 , 6 , 4277 . [26] S. W. Morton , N. J. Shah , M. A. Quadir , Z. J. Deng , Z. Poon ,

P. T. Hammond , Adv. Healthcare Mater. 2014 , 3 , 790 . [27] L. Xie , W. Tong , D. Yu , J. Xu , J. Li , C. Gao , J. Mater. Chem. 2012 , 22 ,

6053 . [28] M. Dash , F. Chiellini , R. M. Ottenbrite , E. Chiellini , Prog. Mater Sci.

2011 , 36 , 981 . [29] A. O. Elzoghby , W. M. Samy , N. A. Elgindy , J. Controlled Release

2012 , 157 , 168 . [30] K. Y. Lee , D. J. Mooney , Prog. Mater Sci. 2012 , 37 , 106 . [31] O. Jeon , D. S. Alt , S. M. Ahmed , E. Alsberg , Biomaterials 2012 , 33 ,

3503 . [32] J. Y. Lim , H. J. Donahue , Tissue. Eng. 2007 , 13 , 1879 . [33] M. O. Klein , A. Bijelic , T. Ziebart , F. Koch , P. W. Kämmerer ,

M. Wieland , M. Knonerding , B. Alnawas , Clin. Implant. Dent. R. 2013 , 15 , 166 .



FULL P

APER


[34] M. Lai , K. Cai , Y. Hu , X. Yang , Q. Liu , Colloids Surf., B. 2012 , 97 , 211 . [35] D. Khang , J. Lu , C. Yao , K. M. Haberstroh , T. J. Webster , Biomaterials

2008 , 29 , 970 . [36] S. Wang , Y. Wan , Y. Liu , Nanoscale 2014 , 6 , 12482 . [37] L. Cao , J. Wang , J. Hou , W. Xing , C. Liu , Biomaterials 2014 , 35 , 684 . [38] B. Li , T. Yoshii , A. E. Hafeman , J. S. Nyman , J. C. Wenke ,

S. A. Guelcher , Biomaterials 2009 , 30 , 6768 [39] M. J. Kim , B. Lee , K. Yang , J. Park , S. Jeon , S. H. Um , D. I. Kim ,

S. G. Im , S. W. Cho , Biomaterials, 2013 , 34 , 7236 .

[40] M. Lai , K. Cai , L. Zhao , X. Chen , Y. Hou , Z. Yang , Biomacromolecules 2011 , 12 , 1097 .

[41] J. C. Wenke , S. A. Guelcher , Expert Opin. Drug Delivery 2011 , 8 , 1555 .

[42] a) E. J. Lee , S. H. Jun , H. E. Kim , H. W. Kim , Y. H. Koh , J. H. Jang , J. Mater. Sci. 2010 , 21 , 207 ; b) Z Xie , X. Liu , W. Jia , C. Zhang , W. Huang , J. Wang , J. Controlled Release 2009 , 139 , 118 ; c) Antoci , G. Harrison , P. Patal , T. A. Freeman , I. M. Shapiro , J. Parvizi , N. J. Hickok , S. Radin , P. Ducheyne , J. Orthop. Res. 2009 , 27 , 701 .



Nanostructured Architectures by Assembling Polysaccharide ...yal310/papers/Wang-2015-Advanced...Adv....

Documents

Transcript of Nanostructured Architectures by Assembling Polysaccharide ...yal310/papers/Wang-2015-Advanced...Adv....