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International Journal of Biological Macromolecules 69 (2014) 464–470

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

abrication of PLLA/�-TCP nanocomposite scaffolds with hierarchicalorosity for bone tissue engineering

ao Lou ∗, Xuejun Wang ∗, Guojun Song, Zheng Gu, Zhen Yangollege of Chemistry, Chemical Engineering, and Environmental Science, Qingdao University, 308 Ningxia Road, Qingdao 266071, China

r t i c l e i n f o

rticle history:eceived 8 February 2014eceived in revised form 19 May 2014ccepted 9 June 2014vailable online 14 June 2014

eywords:LLA

a b s t r a c t

Polymer and ceramic composite scaffolds play a crucial role in bone tissue engineering. In an attempt tomimic the architecture of natural extracellular matrix (ECM), poly(l-lactic acid)/�-tricalcium phosphate(PLLA/�-TCP) nanocomposite scaffolds with a hierarchical pore structure were fabricated by combiningthermal induced phase separation and salt leaching techniques. The nanocomposite scaffold consisted ofa nanofibrous PLLA matrix with a highly interconnected, high porosity (>93%) hierarchical pore structurewith pore diameters ranging from 500 nm to 300 �m and a homogeneously distributed �-TCP nanoparti-cle phase. The nanofibrous PLLA matrix had a fiber diameter of 70–300 nm. The nanocomposite scaffolds

-TCPanoierarchicalone tissue engineering

possess three levels of hierarchical structure: (1) porosity; (2) nanofibrous PLLA struts comprising thepore walls; and (3) �-TCP nanoparticle phase. The �-TCP nanoparticle phase improved the mechanicalproperties and bioactivity of the PLLA matrix. The nanocomposite scaffolds supported MG-63 osteoblastproliferation, penetration, and ECM deposition, indicating the potential of PLLA/�-TCP nanocompositescaffolds with hierarchical porosity for bone tissue engineering applications.

. Introduction

Polymer and ceramic composite scaffolds are currently investi-ated as bone substitutes in the clinical setting [1–4]. One type ofomposite scaffolds combines a polymer matrix with a controlledolume fraction of ceramic particles to enhance the mechan-cal properties and bioactivity [9–13]. Biodegradable syntheticolyesters, such as poly(lactic acid), poly(glycolic acid) and theiropolymer poly(lactide-co-glycolide) are widely used polymers inomposite scaffolds for bone tissue engineering, while commonlysed ceramics include hydroxyapatite, �-tricalcium phosphate andiphasic calcium phosphate [5–8].

In addition to the bioactivity and mechanical properties of thecaffold, the scaffold pore morphology, including fiber size, poros-ty, and pore size, is important for determining cell in-growth14–16]. Ideally, the scaffold surfaces that directly interact withells should possess similar dimensions as the natural extracellularatrix (ECM), which ranges in size from 50 to 500 nm [17,18]. Scaf-

olds that mimic the natural ECM microstructure with nanoscalerchitecture and high surface area improve cell attachment, pro-iferation and differentiation [19–22]. However, the application of

∗ Corresponding authors. Tel.: +86 532 85953796; fax: +86 532 85955527.E-mail addresses: taolou72@aliyun.com (T. Lou), wangxjlt@aliyun.com

X. Wang).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.06.004141-8130/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

nanoscale scaffolds are limited by poor cellular infiltration dueto the pore size of nanoscale scaffolds in the range of several totens of microns. Since cells are also on this scale, it would be dif-ficult for them to traverse the small pores unless the scaffoldsdegraded [23,24]. In general, macropores with a pore diame-ter in the range of 50–300 �m are required for cell penetrationand neo-vascularization of a scaffold in vivo, while microporesin the range of 0.5–10 �m are desired to provide effective deliv-ery of nutrients and physical cues that promote cell functionand fast healing [25–27], therefore, a scaffold with a hierarchi-cal pore structure would be advantageous in tissue constructs. Liuet al. prepared a hierarchically porous bioactive glass scaffold thatdemonstrated potential for tissue in-growth, enhanced bioactivity,and release of ions [28]. Zhang et al. fabricated three-dimensionalpoly(�-caprolactone) scaffolds with a hierarchical pore structure(3–250 �m) that had uniform cell distribution and guided neo-tissue formation [29]. Nanoscale scaffolds with a hierarchicalpore structure should enhance tissue regeneration compared tonanoscale scaffolds with one level of pore structure.

Here, we report the fabrication of three-dimensional poly(l-lactic acid)/�-tricalcium (PLLA/�-TCP) nanocomposite scaffoldswith hierarchical porosity by combining thermally induced

phase separation (TIPS) and salt leaching (SL) techniques. Wehypothesized that these nanocomposite scaffolds would provideimproved mechanical properties and enhanced bioactivity, includ-ing osteoblast proliferation, penetration, and differentiation. We

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T. Lou et al. / International Journal of Bi

haracterized the pore structure and mechanical properties ofhe nanocomposite scaffolds and evaluated the bioactivity of theanocomposite scaffolds with MG-63 osteoblasts compared to aontrol PLLA scaffold.

. Material and methods

.1. Materials

Calcium chloride (CaCl2) (AR, Yantai Sanhe Chem. Co., China)nd sodium phosphate (Na3PO4) (AR, Yantai Sanhe Chem. Co.,hina) were used as starting materials for �-TCP synthesis.oly(ethylene glycol) (PEG) (AR, MW = 10,000, Shanghai Chem. Co.,hina) was used as an additive for forming amorphous Ca phos-hate precursor. PLLA (MW = 56,000) with an inherent viscosityf 1.6 dl/g was purchased from Shandong Medical Appliance Com-any, China. All other reagents and solvents are of analytical gradeAR) and used as received.

.2. ˇ-TCP nanoparticle synthesis

Calcium chloride (CaCl2) (AR, Yantai Sanhe Chem. Co., China)nd poly(ethylene glycol) (PEG) (AR, MW = 10,000, Shanghai Chem.o., China) were dissolved in deionized water to form 0.100 mol/LEG-CaCl2 solution with a PEG/Ca molar ratio of 4. A 0.133 mol/Lodium phosphate (Na3PO4) (AR, Yantai Sanhe Chem. Co., China)olution was prepared in deionized water and added dropwiseo the CaCl2 solution at a Ca/P molar ratio of 1.5. The precipi-ation reaction occurred under stirring at pH 9 and temperatureas controlled at 5 ◦C. The reaction solution was aged for 24 h andas filtered, precipitates were washed thoroughly with deionizedater, and dried at 60 ◦C for 12 h. Finally, the dried precipitatesere calcined at 900 ◦C for 3 h to obtain �-TCP powder [30]. Thehase composition of calcined �-TCP was determined by X-rayiffraction (XRD; Bruker D8 Advance) with Cu K� radiation overhe 2� range of 20–50◦. The particle size was determined using

DTS Zetasizer Nano (Malvern Instruments, Worcestershire, UK)y measuring dynamic light scattering (DLS) of a 10 mg/ml �-TCPuspension in ethanol.

.3. Preparation of PLLA/ˇ-TCP nanocomposite scaffolds

The PLLA/�-TCP nanocomposite scaffolds were fabricated byombining TIPS and SL method, as previously reported [31,32].riefly, a 0.05 g/ml PLLA solution in tetrahydrofuran was pre-ared by heating to 50 ◦C. The �-TCP nanoparticles were addedo the PLLA solution and ultrasonically dispersed in a sonicat-ng bath for 30 min. The mixture was poured into a glass tubediameter = 12 mm) with pre-sieved NaCl particles (50–300 �m),nd frozen at −20 ◦C overnight. The frozen solution was immersednto deionized water for solvent exchange and salt leaching at 4 ◦Cor 7 days. After salt leaching, the samples were refrozen at −20 ◦Cvernight, lyophilized, and cut into 2 mm thick discs.

.4. Characterization of PLLA/ˇ-TCP composite scaffolds

The PLLA/�-TCP nanocomposite scaffolds were imaged withcanning electron microscopy (SEM, JEOL JSM-6390LV) to char-cterize the pore morphology. Samples were sputter-coated withu/Pd at 20 mA for 90 s before imaging.

The nanocomposite scaffold porosity was determined with a

iquid displacement method. Ethanol was used as the displace-

ent liquid since it penetrates easily into the scaffolds. Briefly, theample (weight = W) was immersed in ethanol and placed underacuum to allow the ethanol to penetrate into the scaffold pores.

al Macromolecules 69 (2014) 464–470 465

The scaffold was then removed from the ethanol and immedi-ately transferred to a weighing bottle containing a known initialvolume (V1) and weight (W1) of ethanol. The final volume andweight of the ethanol and the scaffold in weighing bottle was thenrecorded as V2 and W2. The scaffold porosity (ε) is calculated asfollowing: ε = (W2 − W1 − W)/�/(V2 − V1), where the weight differ-ence (W2 − W1 − W) is the weight of the ethanol penetrating intothe scaffold; the volume difference (V2 − V1) is the apparent volumeof the scaffold; and � is the density of ethanol.

The scaffold compressive modulus was tested using a universalelectronic mechanical testing machine (DXLL-1000, Shanghai DejieInstrument Ltd, China) at a crosshead speed of 0.5 mm/min. Thescaffold dimensions were 12 mm diameter and 2.0 mm thickness.Specimens were compressed to ∼50% of their original thickness.The compressive modulus was calculated from the initial slope ofthe stress–strain plot. Five specimens were tested for each sample.

2.5. Cell culture

Samples were sterilized with 70% ethanol and washed thor-oughly with PBS prior to cell seeding. Each scaffold (12 mmdiameter and 2.0 mm thickness) was seeded with 100,000 MG-63 (ATCC) in 50 �L of media and incubated for 2 h to allow cellsto adhere to the scaffolds, then 1 mL of media was added to eachwell. All samples were maintained in Dulbecco’s modified Eagle’smedium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS,Invitrogen) and 1% penicillin–streptomycin (Invitrogen). The sam-ples were cultured in a humidified incubator at 37 ◦C with 5% CO2for 8 days with media changes every two days.

2.6. Cell proliferation

The AlamarBlue colorimetric assay (Invitrogen) was used to ana-lyze MG-63 cell proliferation on the PLLA/�-TCP nanocompositescaffolds following the manufacturer’s instructions. Briefly, afterculture for 3, 5, and 8 days, scaffolds were transferred to new wellplates, washed twice with phosphate buffer saline (PBS) (Invitro-gen), and incubated with 10% (v/v) AlamarBlue in DMEM with 10%FBS for 2 h at 37 ◦C. Triplicate samples were assayed for each con-dition. The assay solution (300 �L) was transferred to an opaque96-well culture plate and fluorescence was read at 560 nm exci-tation and 590 nm emission by a spectrophotometer (SpectraMaxM2e, Molecular Devices). Cell number was determined from therelative fluorescent units using a calibration curve produced bymeasuring the fluorescence of known cell numbers.

2.7. Cell morphology

The cell morphology in PLLA/�-TCP nanocomposite scaffoldswas analyzed by SEM imaging. Briefly, after 8 days of culture,samples were removed from media, rinsed with PBS, and fixedwith Karnovsky’s fixative overnight. After fixing, specimens wererinsed with DI water and dehydrated with sequential incubationsof 20%, 40%, 60%, 80%, 90% and 100% ethanol in DI water for 15 minat each concentration, and then critical point CO2 dried using aHitachi HCP-2 (Hitachi, Tokyo, Japan). Specimens were sputter-coated with Au/Pd at 18 mA for 100 s, and imaged by scanningelectron microscopy (SEM, JEOL JSM-6390LV).

2.8. Histology

For histological analysis, scaffolds were fixed in cold ethanol for10 min, washed with PBS, and then rinsed with DI water. Sampleswere embedded in OCT (Invitrogen) overnight at −20 ◦C. Sections

466 T. Lou et al. / International Journal of Biological Macromolecules 69 (2014) 464–470

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Table 1Porosity of PLLA/�-TCP nanocomposite scaffolds.

PLLA/�-TCP (w/w) Porosity (%)

100:0 95.4 ± 0.3100:10 94.1 ± 0.2100:20 94.7 ± 0.4

Fig. 1. X-ray diffraction pattern of the calcined �-TCP particles.

5 �m) were cut with a cryotome, stained with hematoxylin-eosinH&E, Invitrogen), and visualized by an optical microscope.

.9. Immunohistochemistry

The MG-63 osteocalcin protein deposition was assayed bymmunofluorescent staining after 8-days of cell culture. The sam-les were fixed in 4% methanol-free formaldehyde (Sigma–Aldrich)

n phosphate buffered saline (PBS, Invitrogen) for 15 min, followedy ice-cold PBS washing. The cell membrane was permeabilizedith 0.25 v% Triton X-100 (Sigma–Aldrich) in PBS for 10 min. After

ncubation with 10% goat serum (Abcam, Cambridge, MA) in PBSor 30 min to block non-specific protein binding, the samples werencubated overnight at 4 ◦C in mouse anti-human osteocalcin pri-

ary antibody at a 1:500 dilution in PBS with 0.25 v% Triton X-100.he samples were rinsed three times with PBS, and then incubatedith the secondary antibody (FITC conjugated goat anti-mouse IgG

ntibody (Abcam)) at a 1:500 dilution in PBS for 2 h, rinsed threeimes with PBS, mounted to a coverslip, and the nuclei were stainedith Prolong Gold Antifade reagent with DAPI (Invitrogen). The

amples were imaged with a fluorescent microscope.

.10. Statistical analysis

Statistical analysis was performed using one-way analysis ofariance (ANOVA) followed by Student’s t-test. P values less than.05 were considered statistically significant.

. Results and discussion

.1. ˇ-TCP nanoparticle characterization

The X-ray diffraction pattern of the calcined �-TCP powderhowed peaks at 2� = 25.70◦, 27.77◦, 31.02◦, 32.44◦ and 34.33◦

Fig. 1). The diffraction pattern of the calcined sample matched the-TCP JCPDS 70-2065 card, indicating that the calcined sample is

pure �-TCP phase with high crystallinity. The particle size distri-ution of the �-TCP particles measured by dynamic light scatteringevealed a normal distribution of particle size with a mean diam-ter of 290 ± 32 nm. These results suggest that �-TCP with highrystallinity and uniform particle size was synthesized successfully.

.2. Scaffold pore morphology

The pore morphology of the PLLA/�-TCP (100:40, w/w)anocomposite scaffolds was evaluated with SEM (Fig. 2). The scaf-

olds have a hierarchical pore structure with large pores around

100:30 94.3 ± 0.3100:40 93.8 ± 0.3

50–300 �m in diameter and small pores around 0.5–10 �m indiameter, resulting from the leaching of NaCl particles and ther-mal induced phase separation process, respectively (Fig. 2a and c).The high magnification SEM micrographs show that both large andsmall pores are highly interconnected (Fig. 2b and d). The nanofi-brous PLLA matrix with a 70–300 nm fiber diameter is also clearlyshown. The �-TCP nanoparticles were uniformly dispersed in thePLLA matrix and mainly located at the surface of the organic matrix(Fig. 2b and d). In all, a three-dimensional PLLA/�-TCP nanocom-posite with a hierarchical pore structure was successfully fabricatedby combining TIPS and SL methods.

Control PLLA scaffolds had a continuous nanofibrous matrixwith a 50–300 nm fiber diameter that was similar to the PLLAnanofibrous matrix of the PLLA/�-TCP nanocomposite scaffolds.However, the control PLLA scaffolds have a pore size of 0.5–10 �m,which is much smaller than the PLLA/�-TCP nanocomposite scaf-folds pore size (Fig. 2e and f).

3.3. Porosity

Porosity greatly influenced the physico-chemical properties ofthe scaffolds [33]. Table 1 summarized the measured scaffoldporosity. The nanocomposite scaffolds exhibited slightly decreasedporosity with increased �-TCP nanoparticle content. The largepores (around 50–300 �m diameter) formed by salt leaching con-tributed a major part of scaffold porosity and the nanocompositescaffolds with different �-TCP content had the same amount ofsalt porogen to the volume of PLLA solution. The slight decrease inporosity with increased �-TCP nanoparticle content was due to the�-TCP nanoparticles occupying the small pores (around 0.5–10 �mdiameter) formed during thermal induced phase separation pro-cess. All PLLA/�-TCP nanocomposite scaffolds were highly porous(>93%), even when the �-TCP nanoparticle content reached 100:40(w/w), which could provide more void volume for cell in-growth.

3.4. Compressive modulus

Bone tissue engineering scaffolds should have sufficientmechanical strength for bone regeneration at the site of implan-tation and maintain structural integrity during both in vitro andin vivo cell growth [34,35]. Different concentrations of �-TCPnanoparticles were added to form PLLA/�-TCP nanocompositescaffolds and the compressive modulus of the nanocomposite scaf-folds was measured. The compressive modulus of the PLLA/�-TCPnanocomposite scaffolds improved significantly with increasing�-TCP concentration (Fig. 3). The compressive modulus of the pris-tine nanofibrous PLLA scaffolds was 0.18 ± 0.03 MPa, while thecompressive modulus of the 100:40 w/w PLLA/�-TCP nanocom-posite scaffolds was 0.84 ± 0.07 MPa, a greater than 4-fold increasein modulus compared to the pristine nanofibrous PLLA scaffolds.This increased modulus indicated that incorporation of �-TCPnanoparticles in the nanofibrous PLLA matrix improved mechanical

properties due to particle enhancement. Furthermore, scaffolds ofsingle polymeric material prepared by other methods with higherporosity would result in lower mechanical properties [36,37],but the PLLA/�-TCP nanocomposite scaffolds possessed both high

T. Lou et al. / International Journal of Biological Macromolecules 69 (2014) 464–470 467

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Fig. 2. SEM micrographs of PLLA/�-TCP nanocomposite scaffold

orosity and compressive modulus compared to the pristine PLLAcaffolds.

.5. Cell proliferation

Cell proliferation is a fundamental measure of biocompatibil-

ty and a large cell population is essential for a tissue engineeredone substitute. The proliferation of MG-63 cells in pristine PLLAcaffolds and PLLA/�-TCP nanocomposite scaffolds was evalu-ted with the AlamarBlue assay (Fig. 4). The cell population

:40, w/w) (a–d) and nanofibrous PLLA scaffolds (control) (e, f).

consistently increased on both scaffold types throughout 8-dayculture period. However, the growth rates differed with �-TCP con-centration. MG-63 cell population on the pristine PLLA scaffoldsincreased 4-fold from day 0 to day 8, while the cell populationon the 100:40 w/w PLLA/�-TCP nanocomposite scaffolds increased7-fold over the same time period and reached 0.76 million cells.

These results indicated that the nanocomposite scaffolds pro-vided a suitable microenvironment for cell proliferation and thatthe higher concentration of �-TCP nanoparticles promoted cellproliferation.

468 T. Lou et al. / International Journal of Biological Macromolecules 69 (2014) 464–470

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ig. 3. Compressive modulus of the PLLA/�-TCP (w/w) nanocomposite scaffolds.ata represents the mean ± SD for five samples. P values < 0.05 were considered

tatistically significant (*).

The 70–300 nm fiber diameter nanofibrous PLLA matrix of theLLA/�-TCP nanocomposite scaffolds is similar to natural ECMnd promotes cell adhesion and proliferation [3,24,38]. More-ver, the hierarchical and interconnected pore structure of theanocomposite scaffolds benefits osteoblast penetration and neo-ascularization with 50–300 �m diameter macropores while alsoaintaining an effective nutrient flow and metabolic exchange

or cell proliferation and tissue growth with 0.5–10 �m diametericropores [15,27,39,40]. Introducing �-TCP nanoparticles into the

LLA matrix provides mechanical support, improves the biocom-atibility, and buffers the acidic degradation of PLLA, which wouldlso benefit osteoblast proliferation [5,34,41,42].

.6. Cell morphology

SEM imaging of MG-63 cells cultured on PLLA/�-TCP nanocom-osite scaffolds (100:40, w/w) displayed an elongated shape andpread out discretely within the pores and on the surface of thecaffolds (Fig. 5a and b), signifying cell adhesion and osteogenicctivity. However, MG-63 cells grown on the control PLLA scaffoldsithout hierarchical pore structure were mainly on the surface of

he scaffolds in multiple layers (Fig. 5c). This indicated that hierar-hical pore structures facilitated the cell adhesion, migration, androliferation.

ig. 4. MG-63 proliferation in the PLLA/�-TCP (w/w) nanocomposite scaffolds over days. Each scaffold was seeded with 100,000 MG-63 cells on day 0. Data repre-ents the mean ± SD for three samples. P values < 0.05 were considered statisticallyignificant (*).

Fig. 5. SEM micrographs of MG-63 grown in PLLA/�-TCP (100:40, w/w) nanocom-posite scaffolds (a, b) and PLLA scaffolds (control) (c) after 8 days of culture.

3.7. Histology

Scaffolds were cross-sectioned and H&E stained after 8 days ofculture to evaluate the MG-63 cell penetration. Consistent with theSEM images, MG-63 cells on the PLLA/�-TCP nanocomposite scaf-

folds displayed an elongated shape and spread out within the pores.H&E stained sections of the PLLA/�-TCP (100:40, w/w) nanocom-posite scaffolds demonstrated osteoblast penetration throughoutthe entire scaffold after 8 days culture (Fig. 6a). In contrast, MG-63

T. Lou et al. / International Journal of Biological Macromolecules 69 (2014) 464–470 469

Fig. 6. Images of H&E stained cross-sections of PLLA/�-TCP nanocomposite scaffolds (100:40, w/w) (a) and PLLA scaffolds (control) without hierarchical pore structures (b)a e MG

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fter 8 days of culture. The arrows indicate the top surface of the scaffolds where th

ells cultured in the control PLLA scaffolds without hierarchi-al pore structure (Fig. 6b) tightly adhered to each other and

ormed continuous sheets with multiple layers. The cell penetrationhroughout the PLLA/�-TCP nanocomposite scaffolds suggestedhat the interconnected, hierarchical pore structure maintained an

ig. 7. Immunofluorescent images of PLLA/�-TCP (100:40, w/w) nanocompositecaffolds stained for osteocalcin (green) after 8 days of culture. Nuclei were coun-erstained with DAPI (blue). (For interpretation of the references to colour in thisgure legend, the reader is referred to the web version of the article.)

-63 cells were seeded initially.

effective nutrient flow and metabolic exchange and provided anideal microenvironment for cell in-growth.

3.8. Immunohistochemistry

To investigate the osteoblast functionality and osteogenic activ-ity, the PLLA/�-TCP (100:40, w/w) nanocomposite scaffolds werefurther evaluated with osteocalcin immunostaining. Osteocalcin isa primary non-collagenous protein produced by osteoblasts, whichsignals terminal osteogenic differentiation, and is commonly usedto measure new bone formation [34,42]. Fig. 7 demonstrated thatMG-63 cells (nuclei stained blue) were attached to the pore wallsand osteocalcin (stained green) was observed around the cells inthe PLLA/�-TCP nanocomposite scaffolds. These immunofluores-cence results demonstrated that ECM was deposited and MG-63cells displayed osteogenic activity in the PLLA/�-TCP nanocompos-ite scaffolds.

4. Conclusions

PLLA/�-TCP nanocomposite scaffolds with a hierarchical porestructure were fabricated by combining TIPS and SL techniques.The PLLA/�-TCP nanocomposite scaffolds exhibited high poros-ity; interconnected, hierarchical pore structures; and improved

mechanical strength by introducing �-TCP nanoparticles intonanofibrous PLLA matrix. Tailoring the chemical composition andporous architecture of the scaffolds promoted osteoblast prolifera-tion, penetration, and ECM deposition. Therefore, such biomimetic

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LLA/�-TCP nanocomposite scaffolds show promise for bone tissuengineering applications.

cknowledgments

The authors acknowledge the support from Department of Sci-nce & Technology of Shandong Province (Grant No. 2011YD21025)nd National Natural Science Foundation of China (Grant No.1043001). The authors acknowledge Dr. Stephen J. Florczyk forritical revision of the manuscript and assistance with grammar.

eferences

[1] D. Mohamad Yunos, O. Bretcanu, A.R. Boccaccini, Polymer-bioceramic compos-ites for tissue engineering scaffolds, J. Mater. Sci. 43 (2008) 4433–4442.

[2] H.H. Jin, D.H. Kim, T.W. Kim, K.K. Shin, J.S. Jung, H.C. Park, S.Y. Yoon, In vivoevaluation of porous hydroxyapatite/chitosan–alginate composite scaffolds forbone tissue engineering, Int. J. Biol. Macromol. 51 (2012) 1079–1085.

[3] H. Liu, H. Peng, Y. Wu, C. Zhang, Y. Cai, G. Xu, Q. Li, X. Chen, J. Ji, Y. Zhang,H.W. Ouyang, The promotion of bone regeneration by nanofibrous hydroxyap-atite/chitosan scaffolds by effects on integrin-BMP/Smad signaling pathway inBMSCs, Biomaterials 34 (2013) 4404–4417.

[4] R.A. Perez, J.E. Won, J.C. Knowles, H.W. Kim, Naturally and synthetic smart com-posite biomaterials for tissue regeneration, Adv. Drug Deliv. Rev. 65 (2013)471–496.

[5] X.J. Wang, G.J. Song, T. Lou, Fabrication and characterization of nano-compositescaffold of PLLA/silane modified hydroxyapatite, Med. Eng. Phys. 32 (2010)391–397.

[6] H. Seyednejad, A.H. Ghassemi, C.F. van Nostrum, T. Vermonden, W.E. Hennink,Functional aliphatic polyesters for biomedical and pharmaceutical applica-tions, J. Control. Release 152 (2011) 168–176.

[7] S.V. Dorozhkin, Biphasic, triphasic and multiphasic calcium orthophosphates,Acta Biomater. 8 (2012) 963–977.

[8] J.R. Jones, Review of bioactive glass: from Hench to hybrids, Acta Biomater. 9(2013) 4457–4486.

[9] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biodegradable and bioactiveporous polymer/inorganic composite scaffolds for bone tissue engineering, Bio-materials 27 (2006) 3413–3431.

10] J. Li, H. Sun, D. Sun, Y. Yao, F. Yao, K. Yao, Biomimetic multicomponentpolysaccharide/nano-hydroxyapatite composites for bone tissue engineering,Carbohyd. Polym. 85 (2011) 885–894.

11] G. Daculsi, E. Goyenvalle, R. Cognet, E. Aguado, E.O. Suokas, Osteoconductiveproperties of poly(96l/4d-lactide)/beta-tricalcium phosphate in long term ani-mal model, Biomaterials 32 (2011) 3166–3177.

12] C. Delabarde, C.J. Plummer, P.E. Bourban, J.A. Manson, Biodegradable polylac-tide/hydroxyapatite nanocomposite foam scaffolds for bone tissue engineeringapplications, J. Mater. Sci. Mater. Med. 23 (2012) 1371–1385.

13] J.B. Lee, H.N. Park, W.-K. Ko, M.S. Bae, D.N. Heo, D.H. Yang, I.K. Kwon, Poly(l-lactic acid)/hydroxyapatite nanocylinders as nanofibrous structure for bonetissue engineering scaffolds, J. Biomed. Nanotechnol. 9 (2013) 424–429.

14] L. Indolfi, A.B. Baker, E.R. Edelman, The role of scaffold microarchitecturein engineering endothelial cell immunomodulation, Biomaterials 33 (2012)7019–7027.

15] K.T. Shalumon, K.P. Chennazhi, H. Tamura, K. Kawahara, S.V. Nair, R. Jayaku-mar, Fabrication of three-dimensional nano, micro and micro/nano scaffolds ofporous poly(lactic acid) by electrospinning and comparison of cell infiltrationby Z-stacking/three-dimensional projection technique, IET Nanobiotechnol. 6(2012) 16–25.

16] J.A. Hendriks, L. Moroni, J. Riesle, J.R. de Wijn, C.A. van Blitterswijk, The effect ofscaffold-cell entrapment capacity and physico-chemical properties on cartilageregeneration, Biomaterials 34 (2013) 4259–4265.

17] X. Liu, L.A. Smith, J. Hu, P.X. Ma, Biomimetic nanofibrous gelatin/apatite com-posite scaffolds for bone tissue engineering, Biomaterials 30 (2009) 2252–2258.

[

al Macromolecules 69 (2014) 464–470

18] J.M. Holzwarth, P.X. Ma, 3D nanofibrous scaffolds for tissue engineering, J.Mater. Chem. 21 (2011) 10243.

19] K. Cheng, W.S. Kisaalita, Exploring cellular adhesion and differentiation in amicro-/nano-hybrid polymer scaffold, Biotechnol. Prog. 26 (2010) 838–846.

20] C. Sun, X. Jin, J.M. Holzwarth, X. Liu, J. Hu, M.J. Gupte, Y. Zhao, P.X. Ma, Devel-opment of channeled nanofibrous scaffolds for oriented tissue engineering,Macromol. Biosci. 12 (2012) 761–769.

21] G.A. Saracino, D. Cigognini, D. Silva, A. Caprini, F. Gelain, Nanomaterials designand tests for neural tissue engineering, Chem. Soc. Rev. 42 (2013) 225–262.

22] H.N. Kim, A. Jiao, N.S. Hwang, M.S. Kim, H. Kang do, D.H. Kim, K.Y. Suh,Nanotopography-guided tissue engineering and regenerative medicine, Adv.Drug Deliv. Rev. 65 (2013) 536–558.

23] X.J. Wang, G.J. Song, T. Lou, W.J. Peng, Fabrication of nano-fibrous PLLA scaffoldreinforced with chitosan fibers, J. Biomater. Sci. Polym. E. 20 (2009) 1995–2002.

24] A. Cooper, M. Leung, M. Zhang, Polymeric fibrous matrices for substrate-mediated human embryonic stem cell lineage differentiation, Macromol.Biosci. 12 (2012) 882–892.

25] A.R. Studart, J. Studer, L. Xu, K. Yoon, H.C. Shum, D.A. Weitz, Hierarchicalporous materials made by drying complex suspensions, Langmuir 27 (2011)955–964.

26] D. Khang, J. Choi, Y.M. Im, Y.J. Kim, J.H. Jang, S.S. Kang, T.H. Nam, J. Song, J.W.Park, Role of subnano-, nano- and submicron-surface features on osteoblast dif-ferentiation of bone marrow mesenchymal stem cells, Biomaterials 33 (2012)5997–6007.

27] D. Yoo, New paradigms in hierarchical porous scaffold design for tissue engi-neering, Mater. Sci. Eng. C 33 (2013) 1759–1772.

28] X. Li, X. Wang, H. Chen, P. Jiang, X. Dong, J. Shi, Hierarchically porous bioactiveglass scaffolds synthesized with a PUF and P123 cotemplated approach, Chem.Mater. 19 (2007) 4322–4326.

29] Q. Zhang, Y. Jiang, Y. Zhang, Z. Ye, W. Tan, M. Lang, Effect of porosity on long-term degradation of poly (-caprolactone) scaffolds and their cellular response,Polym. Degrad. Stabil. 98 (2013) 209–218.

30] C. Zou, W. Weng, X. Deng, K. Cheng, X. Liu, P. Du, G. Shen, G. Han, Preparationand characterization of porous beta-tricalcium phosphate/collagen compositeswith an integrated structure, Biomaterials 26 (2005) 5276–5284.

31] X.J. Wang, G.J. Song, T. Lou, Fabrication and characterization of nano compos-ite scaffold of poly(l-lactic acid)/hydroxyapatite, J. Mater. Sci. Mater. Med. 21(2010) 183–188.

32] Q. Zhang, H. Luo, Y. Zhang, Y. Zhou, Z. Ye, W. Tan, M. Lang, Fabrication ofthree-dimensional poly(epsilon-caprolactone) scaffolds with hierarchical porestructures for tissue engineering, Mater. Sci. Eng. C 33 (2013) 2094–2103.

33] D.J. Yoo, Porous scaffold design using the distance field and triply periodicminimal surface models, Biomaterials 32 (2011) 7741–7754.

34] S. Jana, S.J. Florczyk, M. Leung, M. Zhang, High-strength pristine porous chitosanscaffolds for tissue engineering, J. Mater. Chem. 22 (2012) 6291.

35] P.M. Hunger, A.E. Donius, U.G. Wegst, Structure–property–processing correla-tions in freeze-cast composite scaffolds, Acta Biomater. 9 (2013) 6338–6348.

36] S. Bechtle, S.F. Ang, G.A. Schneider, On the mechanical properties of hierarchi-cally structured biological materials, Biomaterials 31 (2010) 6378–6385.

37] S. Bose, M. Roy, A. Bandyopadhyay, Recent advances in bone tissue engineeringscaffolds, Trends Biotechnol. 30 (2012) 546–554.

38] T. Lou, X. Wang, G. Song, Fabrication of nano-fibrous poly(l-lactic acid) scaffoldreinforced by surface modified chitosan micro-fiber, Int. J. Biol. Macromol. 61C(2013) 353–358.

39] H. Jeon, G. Jin, G. Kim, The effect of microsized roughness in nano/microsizedhierarchical surfaces replicated from a lotus leaf on the activities of osteoblast-like cells (MG63), J. Mater. Chem. 22 (2012) 7584.

40] W. Zhang, G. Wang, Y. Liu, X. Zhao, D. Zou, C. Zhu, Y. Jin, Q. Huang, J. Sun,X. Liu, X. Jiang, H. Zreiqat, The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration, Biomaterials 34(2013) 3184–3195.

41] A.J. Wagoner Johnson, B.A. Herschler, A review of the mechanical behavior of

CaP and CaP/polymer composites for applications in bone replacement andrepair, Acta Biomater. 7 (2011) 16–30.

42] Y. Kang, S. Kim, J. Bishop, A. Khademhosseini, Y. Yang, The osteogenic differ-entiation of human bone marrow MSCs on HUVEC-derived ECM and beta-TCPscaffold, Biomaterials 33 (2012) 6998–7007.