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Biomaterials 30 (2009) 2085–2094

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Biomaterials

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Nanostructured biocomposite substrates by electrospinning and electrosprayingfor the mineralization of osteoblasts

Deepika Gupta a,c, J. Venugopal a,b,*, S. Mitra c, V.R. Giri Dev d, S. Ramakrishna a,b,*

a Nanoscience and Nanotechnology Initiative, 2 Engineering Drive 3, National University of Singapore, Singaporeb Division of Bioengineering, 2 Engineering Drive 3, National University of Singapore, Singaporec Amity Institute of Nanotechnology, Amity University, Noida, Indiad Department of Textile Technology, A.C. College of Technology, Anna University, Chennai, India

a r t i c l e i n f o

Article history:Received 30 September 2008Accepted 19 December 2008Available online 23 January 2009

Keywords:ElectrospinningElectrosprayingBiocompositeHydroxyapatiteMineralizationBone tissue engineering

* Corresponding authors. Nanoscience and Nanotneering Drive 3, National University of Singapore, Sinfax: þ65 6773 0339.

E-mail addresses: engjrv@nus.edu.sg (J. VenugRamakrishna).

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.12.079

a b s t r a c t

Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges inbone replacement therapies. Biocomposite nanofibrous scaffolds of poly(L-lactic acid)-co-poly(3-capro-lactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning andelectrospraying techniques in order to create a better osteophilic environment for the growth andmineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped toattain rough surface morphology ideal for cell attachment and proliferation and also achieve improvedmechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size andporosity up to 90% with fiber diameter in the range of 200–700 nm. Nanofibrous scaffolds were char-acterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies oncell–scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HAblended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization andenzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayedbiocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkalinephosphatase activity (ALP) proving that electrospraying in combination with electrospinning producedsuperior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Revolutionary advances in bone tissue engineering methodolo-gies are urgently needed for rising occurrence of bone diseases,accidental damages and defects. Rapid progress in nanotechnologyand its far reaching developments have triggered the use ofnanostructures as scaffolds for the purpose of tissue engineering[1]. As an alternative route to conventional autogenic and allogenictreatments for bone defects, nanofibrous scaffolds have proved tobe convenient and effective in providing mechanical support andosteoconductivity to the growing cells in bone regeneration [2].Even the artificial and metallic bone implants like stainless steelrods, etc. face serious limitations of mismatching mechanical

echnology Initiative, 2 Engi-gapore. Tel.: þ65 6516 4272;

opal), dprsr@nus.edu.sg (S.

All rights reserved.

properties with adjacent tissues as well as causing stress shieldingto the adjoining bone leading to the deterioration of osteoblastfunction of producing new bone material [3]. As a result, nano-fibrous biocomposite scaffolds are being sought after as substratematerials for reinstating large bone defects, possessing uniquesurface and mechanical properties at the nanometer range,mimicking extracellular matrix [4] and eliciting features likebiodegradability, high porosity with large interconnected pores,large surface area [5], enhanced bioactivity, osteoinductivity,adhesion and proliferation of osteoblasts [6].

Electrospun biocomposite nanofibers mimic the compositenature of bone [7] as well as the nanoscale features of extracellularmatrix (ECM) which serves to organize cells and provides signalsfor cellular responses [8]. Nanofibers have been successfullycreated by a simplistic technique of electrospinning wherebya variety of degradable natural and synthetic polymer and ceramicblends can be drawn into nanofibers by applying high voltage andregulating the parameters like polymer concentration, polymerflow rate, distance between the collector and the needle, andapplied voltage [9,10]. Using this approach biocomposite

D. Gupta et al. / Biomaterials 30 (2009) 2085–20942086

nanofibers of Chitosan/HA [11], PCL/Collagen/HA [12], PCL/HA, PCL/Gel/HA [13], Gel/HA [14], PHBV/Hap [15] have been generated forbone tissue engineering. HA is considered the most suitable bio-ceramic for bone replacement therapies as it possesses excellentbioactivity and osteoconductive properties [16,17] but poormechanical stability [18]. Blending of HA with natural and syntheticpolymers has facilitated the formation of HA nanofibers with bettermechanical strength but conversely it may possibly lead to maskingof its osteoinductive property as the HA particles are completelyembedded inside the polymer fiber. In order to achieve betterosteo-induction/conduction, our approach was to electrospray HAnanoparticles on electrospun polymeric nanofibers, simultaneouslythus depositing a uniform layer of HA nanoparticles on fibersurface. Moreover electrospray deposition of HA nanoparticles isadvantageous in producing nanofibrous scaffolds with controlledsurface topography [19]. Like electrospinning, electrospraying alsoinvolves the use of high voltage which stretches the liquid to formconical jet at the tip of the needle and further break it into smallerdroplets due to the applied electric field [20] and can be regulatedby similar parameters.

In this study, biocomposite nanofibrous scaffolds were fabri-cated using poly(L-lactic acid)-co-poly(3-caprolactone) (PLACL),gelatin and HA by blending and spraying methods so as to create anin vitro environment resembling the lowest level of hierarchicalorganization of bone, for the mineralization of osteoblasts. PLACL isa synthetic, biodegradable, non-toxic copolymer of PCL and PLLA

Fig. 1. FESEM images of electrospun biocomposite nanofibers: (a) PLACL (b) P

[21] and has been used as substrate for culturing smooth musclecells [22,23] and endothelial cells [24,25]. Gelatin was used as itoffers integrin binding sites for cell adhesion and carboxylic acidgroups that binds calcium ions present in HA, with ionic inter-actions [26]. This study focused on assessing the impact ofelectrostatic spray deposition of HA nanoparticles compared toblending HA with polymer, on the growth of hFOB cells andtheir mineralization. For comparisons, pure PLACL and PLACL/Gelnanofibers were electrospun and TCP was used as control. Thesenanofibrous scaffolds were evaluated for properties such ashydrophilicity, tensile strength, functional groups, crystallinity,pore size and porosity and mineralization by hFOB cells wasestimated by ARS staining, and SEM images.

2. Materials and methods

2.1. Materials

Human fetal osteoblast cells (hFOB) were obtained from the American TypeCulture Collection (ATCC, Arlington, VA). Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (HAM), fetal bovine serum (FBS), antibiotics and trypsin–EDTA were purchased from GIBCO Invitrogen, USA. Poly(L-lactic acid)-co-poly-(3-caprolactone) (70:30, Mw 150 kDa) was obtained from Boehringer IngelheimPharma, GmbH & Co., Ingelheim, Germany. Gelatin and 1,1,1,3,3,3-hexafluor-2-propanol (HFP) were purchased from Sigma–Aldrich, USA. Dichloromethane(DCM) was obtained from Merck (Germany) and N,N-dimethyl formamide (DMF)was purchased from Sigma Aldrich (USA). CellTiter 96 AQueous One solution waspurchased from Promega, Madison, WI, USA. Alizarin Red-S and cetylpyridinium

LACL/Gel (c) PLACL/Gel/HA (blend) (d) PLACL/Gel/HA (spray) nanofibers.

Table 1Characteristics of biocomposite nanofibers.

Contactanglea

Average fiberdiameter (nm)

Pore size(mm)

Porosity(%)

PLACL 128� 443� 27 3–7 89PLACL/Gel 0� 686� 97 1–7 92PLACL/Gel/HA(b) 55� 198� 107 1–3 88PLACL/Gel/HA(s) 58� 406� 155 2–6 85

a Before plasma treatment. After plasma treatment all surfaces became 100%hydrophilic (0� angle) for nanofibers.

D. Gupta et al. / Biomaterials 30 (2009) 2085–2094 2087

chloride purchased from Sigma, St. Louis, USA. Crystalline hydroxyapatite wasgenerously provided by the Department of Metallurgical and Materials Engineering,Indian Institute of Technology, Chennai, India.

2.2. Fabrication of nanofibrous scaffolds

Solutions of PLACL, PLACL/Gel and PLACL/Gel/HA (blended) were prepared usingelectrospinning method. PLACL was dissolved in DCM/DMF (70:30) to form 10%(w/v) clear solution. PLACL and gelatin were dissolved and mixed in HFP in a ratio of1:1 (w/w) to form 10% (w/v) solution. HA was sonicated for 1 h at an intensity of 40%to form nanoparticular suspension in the solvent. 10% solution of PLACL and gelatinwas made in HFP as noted above in a ratio of 2:1 (w/w) and added to the solution ofHA nanoparticles and left under long stirring, so as to form a 10% (w/v) solution ofPLACL/Gel/HA in the ratio of 2:1:1 (w/w). All solutions were subsequently used forelectrospinning to form random nanofibers. For electrospinning, the polymersolutions of PLACL, PLACL/Gel and PLACL/Gel/HA were separately fed into a 3 mlstandard syringe attached to a 27 G, 25 G and 22 G blunted stainless steel needleusing a syringe pump (KDS 100, KD Scientific, Holliston, MA) at a flow rate of 1 ml/hwith an applied voltage of 19.5 kV, 17.5 kV and 15 kV (Gamma High VoltageResearch, USA), respectively. On application of high voltage the polymer solutionwas drawn into fibers, collected on 15 mm cover slips, spread on collector platewrapped with aluminum foil, kept at a distance of 12–13 cm from the needle tip andsubsequently used for cell culture studies.

For collecting PLACL/Gel/HA fibers by ‘spray’ method a rotating cylinder setupwas used instead of collector plate. The cylinder was wrapped in aluminum foil and15 mm cover slips were stuck on it using double sided tape. PLACL/Gel solution wasfed in one syringe pump with same parameters used for electrospinning while in theother a nanoparticulate solution of 4% HA in methanol was fed at a rate of 2 ml/h andvoltage of 9 kV was provided for spraying HA nanoparticles on the PLACL/Gelnanofibers being formed on the rotating cylinder, simultaneously. These nanofiberswere dried overnight under vacuum and used for characterization and cell culturestudies.

2.3. Characterization of nanofibrous scaffolds

The surface morphology of electrospun nanofibrous scaffolds was studied underField Emission Scanning Electron Microscope (FEI-QUANTA 200F, Netherland) at anaccelerating voltage of 10 kV, after sputter coating with gold (JEOL JFC-1200 fine

Fig. 2. FESEM images of (a) PLACL/Gel/HA (blend) and (b) PLACL/Gel

coater, Japan). Diameters of the electrospun fibers were analyzed from the SEMimages using image analysis software (Image J, National Institutes of Health, USA).Fourier Transform Infrared (FTIR) spectroscopic analysis of electrospun nanofibrousscaffolds was performed on Avatar 380, (Thermo Nicolet, Waltham, MA, USA) overa range of 500–3800 cm�1 at a resolution of 2 cm�1. Hydrophilic nature of theelectrospun nanofibrous scaffolds was measured by sessile drop water contact anglemeasurement using VCA Optima Surface Analysis system (AST products, Billerica,MA). Distilled water was used for a drop formation. Air plasma treatment wasconducted on plain PLACL nanofibrous scaffolds by electrode less radio frequencyglow discharge plasma cleaner (Model: PDC-001, Harrick Scientific Corporation,USA). The samples placed on glass Petri dishes were put in the chamber of plasmacleaner. Plasma discharge was applied to the samples for 1 min with radio frequencypower set as 30 W under vacuum. Pore size of the nanofibrous scaffolds wasmeasured by Capillary Flow Porometer (CFP-1200-A) (Ithaca, NY, USA) by wet-up/dry-up method and the analysis was done using Automated Capillary Flow Poro-meter system software. Porosity of nanofibrous scaffolds was calculated according toHe et al. [27]. Tensile properties of electrospun nanofibrous scaffolds were deter-mined with a tabletop tensile tester (Instron 3345, USA) using load cell of 10 Ncapacity. Rectangular specimens of dimensions 10 mm� 20 mm were used fortesting, at a crosshead speed of 5 mm/min and the data were recorded for every50 ms. The room conditions were controlled at 25 �C and 34% humidity. Tensile stressstrain and elastic modulus were calculated based on the obtained tensile stress–strain curve. X-ray diffraction patterns were observed by SHIMADZU 6000 (Japan)diffractometer. A voltage of 40 kV and a current of 30 mA using a CuKa radiation(l¼ 1.5418), and continuous scan mode with scan speed of 2 �/min was used.

2.4. hFOB cell culture

hFOB cells were cultured in DMEM/F12 media (1:1) supplemented with 10% FBSand 1% antibiotic and antimycotic solutions (Invitrogen Corp, USA) in a 75 cm2 cellculture flask. Cells were incubated at 37 �C in a humidified atmosphere containing5% CO2 for 6 days and the culture medium was changed once in every 2 days. Each ofthe nanofibrous scaffolds on 15 mm coverslip was placed in 24-well plate andpressed with a stainless steel ring to ensure complete contact of the scaffolds withwells. The specimens were sterilized under UV light, washed thrice with phosphatebuffered saline (PBS) and subsequently immersed in DMEM overnight before cellseeding. hFOB cells grown in 75 cm2 cell culture flasks were detached by adding 1 mlof 0.25% trypsin containing 0.1% EDTA. Detached cells were centrifuged, counted bytrypan blue assay using a hemocytometer and seeded on the scaffolds at a density of1.0�104 cells/well and left in incubator for facilitating cell growth.

2.5. Cell morphology studies

Morphological study of in vitro cultured hFOB cells on all scaffolds wereperformed after 15 and 30 days of cell culture, by processing them for SEM studies.The scaffolds were rinsed twice with PBS and fixed in 3% glutaraldehyde for 3 h.Thereafter, scaffolds were rinsed in DI water and dehydrated with upgradingconcentrations of ethanol (50%, 70% 90%, 100%) twice for 10 min each. Final washingwith 100% ethanol was followed by treating the specimens with hexamethyl-disilazane (HMDS). The HMDS was air-dried by keeping the samples in fume hood.

/HA (spray) at high magnification showing surface morphology.

Fig. 3. FTIR analysis of (a) PLACL (b) PLACL/Gel (c) PLACL/Gel/HA (blend) (d) PLACL/Gel/HA (spray) nanofibers.

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Finally, the scaffolds were sputter coated with gold and observed under SEM toobserve the morphology of osteoblast cells.

2.6. Proliferation studies

The cell adhesion and proliferation on the scaffolds were determined using thecolorimetric MTS assay (CellTiter 96 AQueous One solution, Promega, Madison, WI).The reduction of yellow tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium] in MTS to form purple formazancrystals by the dehydrogenase enzymes secreted by mitochondria of metabolicallyactive cells forms the basis of this assay. The formazan dye shows absorbance at492 nm and the amount of formazan crystals formed is directly proportional to thenumber of cells. To process for the MTS assay, samples were rinsed with PBS toremove unattached cells and incubated with 20% MTS reagent in a serum freemedium for a period of 3 h at 37 �C. Absorbance of the obtained dye was measuredat 490 nm using a spectrophotometric plate reader (FLUOstar OPTIMA, BMG labTechnologies).

2.7. Study of alkaline phosphatase (ALP) activity of hFOB cells

ALP activity of hFOB was measured using Alkaline Phosphate Yellow Liquidsubstrate system for ELISA (Sigma Life Sciences, USA). In this reaction, ALP catalyzes

Fig. 4. Stress–strain curve of (a) PLACL (b) PLACL/Gel (c) PLACL/Gel/HA (blend) (d)PLACL/Gel/HA (spray) nanofibers.

the hydrolysis of colorless organic phosphate ester substrate, p-nitro-phenylphosphate (pNPP) to a yellow product, p-nitrophenol, and phosphate.Nanofibrous scaffolds with hFOBs were washed twice with PBS and added with400 ml pNPP liquid to the scaffolds with cells and incubated for 30 min till the colorof solution becomes yellow. The reaction was checked by addition of 100 ml of 2 NNaOH solution following which the yellow color product was aliquoted in 96-wellplate and read in spectrophotometric plate reader at 405 nm.

2.8. Mineralization of hFOB cells

Alizarin Red-S (ARS) is a dye that binds selectively calcium salts and is widelyused for mineral staining. Nanofibrous scaffolds with hFOB cells were washed twicewith PBS and fixed in ice-cold 70% ethanol for 1 h. These constructs were thenwashed twice with dH2O and stained with ARS (40 mM) for 20 min at roomtemperature. After several washes with dH2O, the scaffolds were observed underoptical microscope and images were taken using image software (Leica FW4000,version v 1.0.2), and the stain was extracted with the use of 10% cetylpyridiniumchloride for 1 h and absorbance of the collected dye was read at 540 nm in spec-trophotometer (Thermo Spectronics, Waltham, MA, USA).

2.9. Statistical analysis

All the data presented are expressed as mean� standard deviation (SD) andwere analyzed using Student’s t-test for the calculation of significance level of thedata. Differences were considered statistically significant at p� 0.05.

3. Results and discussion

3.1. Morphology of electrospun nanofibrous scaffolds

Electrospinning technique has gained an up-thrust in recentyears for its ability to produce biomimetic nanofibers for tissueengineering and scaffold characteristics like nanoscale dimensions,high porosity, interconnected pores and large surface area can beattained effortlessly by this technique [28]. SEM micrographs(Fig. 1) of electrospun nanofibrous scaffolds revealed porous,beadless, nano-scaled fibrous structures formed under controlledconditions. PLACL and PLACL/Gel scaffolds showed uniform nano-fibers and interconnected pores with fiber diameter in the range of440� 27 nm and 686� 94 nm respectively (Table 1). Althoughfiber diameter of PLACL/Gel in HFP should have been low due to

Fig. 5. XRD graphs of nanofibrous scaffolds showing HA and polymer peaks.

Table 2Tensile properties of biocomposite nanofibrous membranes.

Nanofibrousmembranes

Tensilestress (MPa)

Tensilestrain (%)

Elasticmodulus (MPa)

PLACL 4.47 111.26 45.6PLACL/Gel 5.89 75.01 99.4PLACL/Gel/HA(b) 2.9 58.9 48.9PLACL/Gel/HA(s) 3.79 80.02 44.4

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high solvent conductivity, high gelatin concentration and solutionviscosity might have played role in production of fiber sizes, inhigher nanometer range. Electrospun PLACL/Gel/HA (blend) nano-fibers showed HA nanoparticles embedded inside the polymer fiberof size 198� 107 nm while in the electrosprayed PLACL/Gel/HAnanofibers, the HA nanoparticles were sprayed uniformly on andaround the fibers thus forming a layer of HA on surface of thepolymer fibers, increasing fiber diameter to 406�155 nm. HAnanoparticles were polydispersed as some amount of aggregation

Fig. 6. MTS assay for hFOB cells proliferation on biocomposite nanofibrous scaffolds andproliferation as compared to control obtained by t-test. (*) represents p� 0.05 (b- stands fo

might have occurred and larger particles sat between interfiberspaces while smaller ones tend to stick on the fiber surface,imparting a rough texture to the scaffolds (Fig. 2). According toMartinez et al. [29] surface roughness of nanofibrous scaffolds isdesirable for better cell attachment and growth which is alsoenhanced by the presence of functional groups and surfacehydrophilicity [30]. Functional groups present in nanofibers wereanalyzed using FTIR (Fig. 3) in which characteristic bands of gelatinwere observed in both PLACL/Gel/HA (blend and spray) and PLACL/Gel scaffolds where typical N–H stretch for amide A and C–Hstretch for amide B were obtained at 3317 cm�1 and 3109 cm�1

respectively, suggesting the presence of NH2 functional groups ontheir surface. Amide bands of gelatin, similar to the amide I–IIIpeaks from protein constituents of bone (i.e. Type I collagen) [31]were also obtained for these scaffolds where amide I band for C]Ostretch, amide II peak for N–H bend coupled with C–N stretch andamide III peak for N–H bend pertaining to triple helical structure ofgelatin were obtained at 1655 cm�1, 1558 cm�1 and 1275 cm�1,respectively. Stretching vibration for PO4

3� group from mineral HAwas obtained at 1096 cm�1 for P–O stretch and at 573 cm�1 for P–Ostretch coupled with P–O bend, with sharper peak for HA sprayednanofibers and bands pertaining to CO3

2� group from carbonatesubstituted OH and PO4

3� groups in HA were obtained at about1400 cm�1, in both the HA containing scaffolds, which is similar tothat found in bone [31,32], thus confirming bone like chemicalcomposition of the PLACL/Gel/HA (blend and spray) scaffolds.Bands related to OH group in the region 3500–2500 cm�1 of spectrawere found in gelatin containing scaffolds suggesting theirhydrophilicity.

Contact angle data (Table 1) supported the fact that incorpora-tion of gelatin made scaffolds are hydrophilic as compared to plainPLACL scaffolds that were found to be highly hydrophobic withcontact angle of 128� � 3. The hydroxyl groups present in gelatinforms hydrogen bonds with water molecules thus imparting therelevant hydrophilicity. Following plasma treatment, hydrophobicscaffolds were found to be 100% hydrophilic. Liston et al. reportedthat air plasma produce polar groups that modify the surfaceenergy, making it more reactive towards water molecule [33]. Largepore size and porosity, as high as 90%, is desirable as it allowsnutrients and waste flow and also provides enough room forregenerating ECM [34]. Pore sizes and porosity for the scaffolds

TCP. Bar represents mean standard deviation. Asterisks indicate significance level ofr ‘blend’ and s- for ‘spray’).

Fig. 7. ALP activity of hFOB cells on biocomposite nanofibrous scaffolds and TCP. Bar represents mean standard deviation. Asterisks indicate significance level of proliferation ascompared to control obtained by t-test. (*) represents p� 0.05 and (**) represents p� 0.001.

D. Gupta et al. / Biomaterials 30 (2009) 2085–20942090

ranged between 1 and 7 mm and 85–92% respectively, showing thescaffolds to be highly porous in structure (Table 1). Pore sizeobtained for PLACL/Gel/HA (s) was more than PLACL/Gel/HA (b)(Table 1) which could be due to the difference in fiber diameters

Fig. 8. SEM micrographs showing morphology and mineralization of hFOB cells on nanofibr(d) PLACL/Gel/HA (spray) nanofibers (e) TCP.

which effects the spacing between fibers and subsequently the poresize. According to Li et al. decrease in fiber size of PLA nanofibrousmembrane, decreases the pore diameter [35]. Nanofibers showeda typical non-linear stress–strain curve as illustrated in Fig. 4.

ous substrates after day 15 of culture. (a) PLACL (b) PLACL/Gel (c) PLACL/Gel/HA (blend)

Fig. 9. SEM micrographs showing mineralization by hFOB cells after day 30 of culture. (a) PLACL (b) PLACL/Gel (c) PLACL/Gel/HA (blend) (d) PLACL/Gel/HA (spray) nanofibers (e) TCP.

D. Gupta et al. / Biomaterials 30 (2009) 2085–2094 2091

Comparing the mechanical properties of nanofibers, PLACL/Gel/HA(sprayed) scaffolds showed tensile strain to be between pure PLACLand PLACL/Gel. Higher tensile stress and strain values wereobtained for PLACL/Gel/HA (spray) scaffolds than PLACL/Gel/HA

Fig. 10. Quantification of mineral deposition in hFOB cells by the method of Alizarin Red-Sobtained by t-test. (*) represents p� 0.05 and (**) represents p� 0.001.

(blend) scaffolds which could be attributed to the fact that blendinginvolves physical mixing of mechanically mismatched materials,while spraying results in superficial dispersion of HA nanoparticles,hence not much affecting the intrinsic property of polymer. Elastic

staining. Asterisks indicate significance level of proliferation as compared to control

Fig. 11. Light microscopy images of ARS staining for the mineralization in hFOB cells for day 15 and 30. (a) PLACL (b) PLACL/Gel (c) PLACL/Gel/HA (blend) (d) PLACL/Gel/HA (spray)nanofibers (e) TCP.

D. Gupta et al. / Biomaterials 30 (2009) 2085–20942092

D. Gupta et al. / Biomaterials 30 (2009) 2085–2094 2093

modulus of both blended and sprayed scaffolds were nearing toeach other which showed that both the scaffolds were equallyefficient in resistance to deformation. Fabrication technique of thesprayed scaffolds (use of rotating drum setup) could be one ofthe reasons for better tensile properties of sprayed scaffolds thanthe blended scaffolds since due to rotation, sprayed scaffolds hadmore oriented fibers hence better mechanical strength [25]. Acomparison of tensile properties of nanofibers with respect totensile stress strain and elastic modulus are illustrated in Table 2.XRD patterns (Fig. 5) of electrospun nanofibers showed two broadpeaks corresponding to PLACL, since it is a co-polymer of PLLA andPCL. The first peak was obtained at 2q of 16.42� for PLLA, which wassimilar to the peak obtained by Tsuji et al. [36] and second at 22.28�

pertaining to PCL. Characteristic peaks for HA were obtained at25.72�, 28�, a broad and intense peak from 30� to 34�, 39.7� and 46�

of 2q. HA containing scaffolds expressed peaks corresponding to HAat 28� and 30�–34� with higher intensity peaks in HA sprayedscaffolds owing to fully exposed HA particles concentrated on thesurface unlike PLACL/Gel/HA blended scaffolds.

3.2. Cell–scaffold interaction and mineralization

Scaffold properties play a pivotal role in controlling the cellgrowth and morphology and impose a direct influence on intra-cellular responses. Cell behavior such as adhesion, spreading andproliferation represent the initial phase of cell–scaffold communi-cation that subsequently effect differentiation and mineralization[13]. In this study, effect of scaffold composition and surfacedeposition of HA were analyzed on cell morphology, growth andphenotypic expression of hFOB cells. Results for MTS studies (Fig. 6)showed a significant increase (p� 0.05) for day 10 and 15 of cellculture, in the proliferation of hFOB cells on both blended and sprayPLACL/Gel/HA scaffolds as compared to PLACL, PLACL/Gel and TCP.On day 15, increase in proliferation of cells was 78% and 89%respectively, for PLACL/Gel/HA blended and sprayed scaffolds whileon PLACL and PLACL/Gel scaffolds only 52% and 41% increase wasseen, respectively. Furthermore, a significant increase in prolifera-tion (p� 0.05) was seen on PLACL/Gel/HA (sprayed) scaffolds ascompared to PLACL/Gel/HA (blended) scaffolds on day 15 of culture.Alkaline phosphatase is a membrane bound enzyme and its activityis used as an osteoblastic differentiation marker [37], as it isproduced only by cells showing mineralized ECM [38]. Results ofALP activity (Fig. 7) of hFOB cells were significantly (p� 0.001)enhanced on PLACL/Gel/HA (spray) scaffold as compared to HA(blended) scaffold on day 15 and this time point marked the onsetof mineralization in hFOB cells. Similar results were obtained byJones et al. [39] where the ALP activity for human osteoblast cells(HOB) was highest on day 14 after which a decrease was obtaineddue to the beginning of mineralization in HOB. Lesser activity onPLACL/Gel scaffolds can be attributed to the degradation of gelatin,which also implies that gelatin degradation, was slowed down inPLACL/Gel/HA scaffolds by integration of HA, thus showing higheractivity. The SEM images of hFOB (Fig. 8) revealed normal cellmorphology on all scaffolds with the formation of mineral particleson the surface of cells after day 15 of cell culture. After day 30 ofculture, more mineralization was seen with fused cells forminga thick layer on the surface of the scaffolds along with ECMproduction by cells (Fig. 9). Mineralization refers to cell-mediateddeposition of extracellular calcium and phosphorus salts whereanionic matrix molecules take up the Ca2þ, phosphate ions andserve as nucleation and growth sites leading to calcification [40].Mineralization was quantified by ARS (Fig. 10) studies, showingdegree of mineralization obtained on day 15 and 30 of cell culturewas in the order of PLACL/Gel/HA (spray)> PLACL> PLACL/Gel/HA(blend)> TCP> PLACL/Gel and percentage increase was 76%, 66%,

66%, 58% and 48% respectively. The HA sprayed scaffold showed50% higher mineralization than HA blended scaffold with signifi-cant level of p� 0.001 on day 30 of cell culture confirming theeffectiveness of spray deposition of HA on fibers in mineralformation by hFOB. Optical microscopy images (Fig. 11) of scaffoldsstained with ARS for days 15 and 30 supported the above datawhere high intensity of staining minerals was observed on HAsprayed scaffolds displaying an overall enhancing effect in miner-alization of hFOB cells and proving electrospraying as an indis-pensable method for the fabrication of superior nanofibrousscaffolds for bone tissue engineering.

4. Conclusion

Electrospraying of HA nanoparticles in combination with elec-trospinning of fibers offered favorable surface topography andosteophilic environment for the attachment and growth of hFOBcells. Due to the complete exposure of HA nanoparticles, theirosteoconductive and osteoinductive effect was enhanced leading tobetter osteoblast response and expression of mineralization. Inconjugation with high surface area provided by nanofibers, pres-ence of HA nanoparticles augmented the total available surface areafor cell attachment. Moreover, PLACL/Gel/HA (spray) nanofibrousscaffolds showed suitable mechanical properties sufficient forsupporting hFOB cells. Though all nanofibrous scaffolds supportedhFOB cells, their proliferation, ALP activity and mineralization werenotably more on PLACL/Gel/HA (spray) scaffolds, qualifying them tobe appropriate for growth and mineralization of osteoblasts forbone tissue engineering.

Acknowledgements

This study was supported by the Office of Life Sciences in theNational University of Singapore and StemLife Sdn Bhd, 50450Kuala Lumpur, Malaysia.

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