Functionally graded β-TCP/PCL nanocomposite scaffolds: In vitro evaluation with human fetal...

Functionally graded b-TCP/PCL nanocomposite scaffolds:In vitro evaluation with human fetal osteoblast cells forbone tissue engineering

Seher Ozkan, Dilhan M. Kalyon, Xiaojun YuDepartment of Chemical, Biomedical and Materials Engineering, Stevens Institute of Technology, Hoboken,New Jersey 07030

Received 20 September 2008; accepted 9 December 2008Published online 18 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32425

Abstract: The engineering of biomimetic tissue relies onthe ability to develop biodegradable scaffolds with func-tionally graded physical and chemical properties. In thisstudy, a twin-screw-extrusion/spiral winding (TSESW)process was developed to enable the radial grading of po-rous scaffolds (discrete and continuous gradations) thatwere composed of polycaprolactone (PCL), b-tricalcium-phosphate (b-TCP) nanoparticles, and salt porogens. Scaf-folds with interconnected porosity, exhibiting myriadradial porosity, pore-size distributions, and b-TCP nano-particle concentration could be obtained. The results of the

characterization of their compressive properties andin vitro cell proliferation studies using human fetal osteo-blast cells suggest the promising nature of such scaffolds.The significant degree of freedom offered by the TSESWprocess should be an additional enabler in the quest to-ward the mimicry of the complex elegance of the nativetissues. � 2009 Wiley Periodicals, Inc. J Biomed Mater Res92A: 1007–1018, 2010

Key words: scaffold; tissue engineering; functionallygraded; nanocomposite; osteoblast; extrusion

INTRODUCTION

Tissue engineering involves in vitro culturing ofhuman cells on a biodegradable and preferably bio-resorbable scaffold which are then implanted intothe human body, where the cells continue to prolif-erate and differentiate while the underlying scaffoldmaterial degrades.1 This exciting multidisciplinaryfield is challenged by the complex gradations in celltype and extra cellular matrix, microstructures foundin native tissues.2–6 To meet this challenge tissue en-gineering has evolved accordingly by focusing onthe necessity of developing functionally graded scaf-folds. Such scaffolds are graded in composition andstructure to guide the resulting cell/material interac-tions, cell and nutrient penetration, mechanical anddegradation properties, and the spatial distributionand temporal coordination of biochemical signals.These properties affect cell adhesion, migration, pro-liferation, and differentiation necessary for the gener-ation of the gradations found in native tissue.2–6

In the area of bone tissues, the available approachesfor functional grading of scaffolds include compres-sion molding of differentially compacted powders,7

casting of differential polymeric cement layers,8 stack-ing and bonding of solvent-casted and micro-moldedporous polymer films, stacking and suturing of elec-trospun woven meshes,9–12 centrifugation,13 andsupercritical CO2 foaming.14 Such approaches7–14 relyheavily on user skills for reproducibility and consis-tency. Other methods include rapid prototyping,fused deposition modeling, and 3D fiber deposi-tion.15–17 These fabrication methods can be handi-capped in terms of the biomaterials that they can pro-cess and simultaneous control of differentials in com-positions and geometries. A recently introducedhybrid extrusion and electrospinning method allowsthe grading of nonwoven meshes of fibers and nano-fibers, but only applies to compositions that can beelectrospun.18,19

In the current study, a twin-screw-extrusion/spiralwinding (TSESW) process, suitable for the fabrica-tion of relatively large scaffolds with gradations inporosity, pore size, chemical composition, and me-chanical properties, is reported. Its abilities for gen-erating radially varying compositional and porositygradations in tubular scaffolds are demonstrated

Correspondence to: D. M. Kalyon; e-mail: [email protected]

� 2009 Wiley Periodicals, Inc.

using polycaprolactone (PCL), incorporated with b-TCP nanoparticles. The effects of the gradations ofpore size, porosity, and b-TCP concentrations on thedevelopment of compressive properties are eluci-dated. The biocompatibility of the functionallygraded scaffolds and suitability for bone tissue engi-neering are assessed with an in vitro study employ-ing human fetal osteoblast cells (hFOB).

EXPERIMENTAL

The TSESW process for scaffold fabricationand materials

The TSESW process integrates the twin screw extrusionprocess with a modified filament winding method (desig-nated here as ‘‘spiral winding’’). The twin-screw-extrusionpart of the process enables the carrying out of multipleunit operations including solids conveying, melting, tem-perature control, distributive, and dispersive mixing ofparticles and nanoparticles, and deaeration and shaping ofthe biocomposite. The feeding of the solid and liquidingredients in a time-dependent fashion further generatesspatial gradations in composition and porosity of theextrudates, which are subsequently wound around a man-drel that concomitantly rotates and translates sidewayscreating a helical and spiral trajectory (Fig. 1).

To fabricate radially and/or axially graded scaffolds thetypes and feed rates of the ingredients, the sizes of par-ticles and nanoparticles, and the mandrel rotation andtranslation (in the transverse to extrusion direction) speedscan be manipulated. The parameters of the twin-screw-extrusion and the motion of the mandrel involved in theTSESW process can be controlled leading to accurate and

reproducible scaffold dimensions and compositional distri-butions. Furthermore, the TSESW method is amenable tolarge-scale manufacturing and suitable for handling poly-mers, solutions and gels under different temperature con-ditions, for example, wet or melt extrusion.

A 7.5-mm mini twin-screw extruder (Material Processingand Research, Hackensack, NJ) with a rectangular slit die(width 10 mm and gap 1 mm) was used in conjunction witha mandrel with a diameter of 1.5 mm, placed orthogonal tothe extrusion direction. [Fig. 1(a)]. The twin-screw extruderhas the capability to control temperatures over multiplezones, a feature which is generally not possible with con-ventional and rapid prototyping/fiber deposition type scaf-fold manufacturing methods. A rotating mandrel is placedat an angle to the extrusion direction to allow the windingof the extrudate on the mandrel surface [Fig. 1(a)]. Themandrel is rotated as well as translated in the transverse toextrusion direction to control the helix angle of the trajec-tory of the extrudate, wound around the mandrel, and toset the thicknesses of the successive layers of the scaffold.

Process parameters such as, polymer/solvent ratio,porogen particle size, and modality and screw RPM wereselected so that for each combination of concentrations ofnano b-TCP and porogen, the clogging of the die is pre-vented and the extrudate holds its shape upon exiting thedie. The inner and outer diameters and the length of thescaffolds were kept at 1.5, 8, and 100 mm, respectively.Sample designations of fabricated scaffolds and their vari-ous properties are given in Table I. Inner diameter of thescaffold can be decreased or increased to above or belowthe 1.5 mm diameter of this study by simply decreasing orincreasing the mandrel diameter, respectively. There is noupper limit to the outside diameter of the scaffold.

In our experiments, PCL pellets (Mn 80,000, SigmaAldrich, Milwaukee, Wisconsin) were dissolved indichloromethane (DCM; Pharmco) and were fed to the ex-

Figure 1. Twin screw extrusion spiral winding process, TSESW (a) Typical scaffolds (b-e) Scale bar represents 2 mm.[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

1008 OZKAN, KALYON, AND YU

Journal of Biomedical Materials Research Part A

truder with a syringe pump. A second syringe pump wasused to feed the b-tricalciumphosphate (b-TCP) bioceramicnanoparticles (Sigma) incorporated into a second stream ofPCL/DCM solution. Nano b-TCP particles were used asthey were purchased and SEM imaging of unprocessedpowder showed that there is a broad particle size distribu-tion with the sizes in the 20 nm to 5 lm range. Two loss-in-weight solids feeders (available from Brabender and K-Tron) were used for the feeding of two NaCl powderstreams (Sigma Aldrich) with two different particle sizeranges (250 to 500 lm and 3 to 20 lm). The targeted poresizes were selected to demonstrate the possibility of gener-ating a broad range of pore sizes to enable the study ofthe effects of pore size on mechanical properties.

Eleven sets of homogeneous or functionally-graded scaf-fold samples were fabricated to enable the investigation ofthe effects of porosity, spatial distributions of pore size,and material compositions on mechanical properties(Table I). In some experiments the feed rate of 250 lmNaCl crystals was adjusted and scaffolds with differentporosity values changing from 60 to 75% were manufac-tured (these scaffolds are designated as PCL1-4). Anothergroup of samples involved a bimodal mixture of porogenparticles (20% 20 lm and 80% 250 lm) to increase the po-rosity and enhance the interconnectivity of the pores of thescaffold (PCL5). Scaffolds with the same porosity but dif-ferent pore sizes at different layers were also fabricated(PCL6-8). Besides the three-layered scaffolds with poresize gradients, b-TCP/PCL nanocomposite scaffolds withconcentration gradients of b-TCP were also generated(PCL-TCP1 and 2). After extrusion, the extrudates weredried for 48 h to remove their DCM solvent. Followingdrying the extrudates were kept in deionized, DI, waterfor 48 h to leach out the porogen. During leaching, DIwater was replaced every 8 h.

Scaffold characterization

Thermo-gravimetric analysis (TGA) was used to deter-mine the decomposition temperature and compositions ofthe ingredients (TGA-Q100, TA Instruments). In TGA thescaffold samples were heated from 25 to 5508C at a heatingrate of 158C/min under a nitrogen environment. The poros-

ity, [, values of the tubular PCL and PCL/ b-TCP scaffoldswere estimated on the basis of the scaffold geometry, theapparent density values of the scaffolds, and the densitiesof the ingredients. The estimated porosity values were veri-fied using Hg intrusion porosimetry using the filling pres-sure range of 0–420 MPa (Poremaster 60, QuantachromeInstruments, Boynton Beach, FL). The porosity values weredetermined from the total Hg intrusion volumes, Vi:

2 ¼ Vi=ðVi þ 1=qTRUEÞ ð1Þ

where qTRUE is the density of the porous scaffold measuredusing He pycnometry (AccuPyc 1330, Micrometrics, Nor-cross, GA).

A differential scanning calorimeter (DSC-Q50, TAInstruments) was used for the measurement of the bulkcrystallinity values of the scaffolds. Sample weights werein the 5–10 mg range. In the DSC experiments, the scaffoldsamples were scanned from 0 to 1508C at a heating rate of158C/min, using nitrogen as purge gas. The degree ofcrystallinity, Xc, values of the scaffolds were determinedfrom the ratio of the heat of fusion, DHf, of the PCL deter-mined after the first heating of the scaffold sample to thatof pure crystalline PCL, DHc, which is 136 J/g.20

Xc ¼ DHf=DHc ð2Þ

For the heat of fusion determination of the scaffold sam-ples, only the net weight of the PCL was considered, thatis, the weight of b-TCP was subtracted from the totalweight of the sample.

A LEO Gemini 982 scanning electron microscopeequipped with a field emission column was employed forthe imaging of the scaffold samples prior to and after theculturing of cells with acceleration voltages ranging from 1to 15 kV. Some of the scaffolds were dipped into liquidnitrogen for 5 minutes and then fractured in the transverseto flow direction in order to expose the pore morphologyand interconnectivity. The samples that were used for theimaging of b-TCP nanoparticles inside the polymer matrixwere not coated with gold to avoid confusion with sput-tered gold particles. In such cases a relatively low 1 kVacceleration voltage was used. The rest of the sampleswere sputter coated with about 20 nm of Au (using a SPIModule sputter coater).

TABLE IScaffold Designations and Their Pore Size and Layer-by-Layer Compositions

ScaffoldDesignation

Mean Pore Size, lm b-TCP: PCL Ratio

Inner Layer Middle Layer Outer Layer Inner Layer Middle Layer Outer Layer

PCL1 250 250 250 0:100 0:100 0:100PCL2 250 250 250 0:100 0:100 0:100PCL3 250 250 250 0:100 0:100 0:100PCL4 250 250 250 0:100 0:100 0:100PCL5 250/20 250/20 250/20 0:100 0:100 0:100PCL6 250 250 20 0:100 0:100 0:100PCL7 20 N/A 250 0:100 0:100 0:100PCL8 20 250 20 0:100 0:100 0:100PCL9 20 20 20 0:100 0:100 0:100

PCL_TCP1 250 250 250 3:97 3:97 3:97PCL_TCP2 250 250 20 3:97 3:97 48:42

FUNCTIONALLY GRADED �-TCP/PCL NANOCOMPOSITE SCAFFOLDS 1009


Compression tests following ASTM 695-02a were carriedout at a crosshead speed of 0.02 mm/s. A Bose EnduratecELF 3200 compression tester with a 0–250 N load cell wasused. Compression samples were 7 to 8 mm in outer diam-eter, 1.5 mm in inner diameter and 14 to 16 mm in length.The compressive stress versus strain data were analyzedfrom load-displacement measurements. The modulus ofelasticity and compressive yield strength values weredetermined according to ASTM 695-02a.

Interconnectivity of the scaffolds was assessed by thedrop-by-drop deposition of a dye, that is, propidiumiodide/tween20 at a single location on the surface of thescaffold. The deposition of the dye was continued until theentire surface of the scaffold changed color. The scaffoldwas sectioned with a scalpel to image the extent of dyepenetration throughout the scaffold.

Cell-scaffold interactions

To evaluate the biocompatibility of the scaffolds fabri-cated by TSESW method, a cell-culturing study was carriedout using only the PCL4 type scaffold specimens ([ 5 0.75).About 100-mm long scaffold specimens were divided into5-mm long sections. They were sterilized upon immersioninto 70% ethanol for 2 h. The scaffolds were then washedwith phosphate buffered saline (PBS) several times toremove the ethanol, followed by exposure to UV light for45 min following Yu et. al.21 The sterilized scaffolds werekept in complete growth medium until cell culturing.

Human fetal osteoblast (hFOB) cells were used for theassessment of the biocompatibility of the scaffolds. The hFOBcells were selected for culturing due to their relatively highgrowth rates and rapid differentiation into mature osteo-blasts.22–28 HFOB cells, which were obtained from AmericanType Culture Collection (ATCC-designation hFOB 1.19),were passaged once a week in T-75 culture flasks and cul-tured in complete growth medium containing a 1:1 mixtureof Ham’s F12 medium (GIBCO) and Dulbecco’s modifiedEagle’s medium without phenol red with 2.5 mM L-gluta-mine, 0.3 mg/mL of Geneticin (G418), 10% fetal bovine se-rum, 100 lg/mL penicillin, and 100 mU/mL streptomycin(ATCC). Cultures were grown at 378C in a humidified 5%CO2 atmosphere. Media were changed every 2 days and cul-tures were passaged with 2.5 g/L trypsin (mass fraction0.25%) and 1 mmol/l EDTA (Gibco, Invitrogen Corporation).After six passages in complete growth medium, cultures of90% confluent cells were trypsinized, washed with PBS(ATCC) and centrifuged at 800 rpm for 4 minutes. Centri-fuged hFOB cells were resuspended in fresh complete growthmedium. Following formation of a dispersed cell suspension,cells were countedwith a hemocytometer. 200 lL cell suspen-sion containing 105 cells was seeded onto the external and in-ternal surfaces of the PCL scaffold using a micropipette. Thescaffolds were flipped a number of times during seeding. Af-ter waiting 4 h to allow the hFOB cells to attach onto the scaf-fold surfaces, 1 mL of complete growth medium supple-mented with 50 lM ascorbic acid (Sigma), 10 mM b-glycero-phosphate (Sigma), and 100 nM dexamethasone (Sigma;differentiationmedium)was added into eachwell.

The number of cells attached to the surfaces of the scaf-folds 24 h later was determined to be about 50,000 cells/

scaffold. The scaffolds were incubated for 8 days. Differen-tiation medium was replaced every 2 days.

Cell viability and proliferation

The number of viable cells on the scaffolds was deter-mined using the MTS assay (CellTiter96TM AQueousAssay, Promega), following the CellTiter96TM applicationprocedure. Assay medium was transferred into a 96-wellassay plate and the absorbance was measured at 490 nmusing a SYNERGY HT plate reader (KC4, BIO-TEK).

Alizarin red calcium quantification

Mineralized matrix synthesis of hFOB cells on the scaf-fold specimens was analyzed using the Alizarin Red stain-ing method for Ca2þ deposition.21,29 The scaffold speci-mens were fixed with 4% paraformaldehyde at 48C for 1 hand then stained with 10% Alizarin Red (Sigma) solutionfor 10 min. After washing five times with distilled water,the red matrix precipitate was solubilized in 10% cetylpyri-dinium chloride (Sigma), and the optical density of the so-lution was read at 562 nm with a SYNERGY HT platereader (KC4, BIO-TEK).

Cell morphology and penetration depth by SEM,optical microscopy and confocal microscopy

Tissue constructs obtained upon the incubation of thehFOB cells were removed, washed with PBS, and werefixed with 4% paraformaldehyde (mass fraction) in PBS atroom temperature for 30 min. Upon fixation the constructswere washed again, followed by staining using methyleneblue (Aldrich) for stereo-microscopy and using propidiumiodide (Invitrogen) for confocal microscopy. A LSM 510confocal laser microscope equipped with a HeNe1 542 nmlaser was used to image cells at different locations of thescaffold to elucidate cell penetration patterns. Some of theconstruct samples were prepared for SEM analysis upondehydration in graded ethanol changes after fixation,washing, and drying at room temperature.

Data analysis

At least three samples per condition were used to mea-sure the bulk crystallinity, composition, and mechanicaltesting. The MTS assay experiments were repeated twice.Three samples per condition were used for each run. Alldata are reported in terms of 695% confidence intervals,determined according to Student’s-t distribution.

RESULTS AND DISCUSSION

Structural and morphological characteristicsof the scaffolds

The different types of radially graded scaffoldsgenerated using the TSESW process are shown in



Figure 1(b). Scaffolds with continuous grading orscaffolds with discrete layers (multilayered) withstep changes in the pore size and/or b-TCP nano-particle concentrations could be obtained [Fig. 1(c-e)].The mean pore size and bulk crystallinity values ofthe different types of scaffolds are given in Table II.The porosity values were altered in the 60–78%range. The differences in the crystallinity of the PCLfound in different scaffolds were determined to benegligible (Table II).

Figure 2(a) shows the typical SEM micrographsof cross-sections of scaffolds of type PCL-TCP2(Table I) with larger pores at the core and smallerpores at the skin layer. TCP nanoparticle clustersform at the higher concentration [see the top inset ofFig. 2(a)]. The concentration versus radial distancedistribution of b-TCP nanoparticles in PCL-TCP2scaffold as determined with TGA is shown in Figure2(b). The b-TCP nanoparticle concentration increasesfrom 2.8 to 48 (weight, w, %) going from the inner

to the outer surface of the PCL-TCP2 scaffolds. Theability to generate such complex radial distributionsof nanoparticles is one of the basic features of theTSESW technology.

Figure 3 shows the typical SEM images of PCL7and PCL8 type multilayered scaffold samples, whichwere cryo-fractured in the transverse to flow direc-tion. The layer thickness and porosity are uniformalong the extrusion direction throughout the scaf-fold. The availability of homogeneously distributed,highly concentrated solid particles prior to leachingshould be instrumental in the prevention of the col-lapse of the wet polymer upon extrusion. All layerspreserved the targeted properties and dimensionsfor each layer and the intermingling of the differentlayers was not observed.

The interconnectivity (open-celled type) and sur-face topographies of scaffold samples of type PCL4,PCL5, and PCL9 are shown in Figure 4. These micro-graphs reveal well-interconnected structures [Fig.4(a-i)] with porosity levels surpassing 70%. No signsof salt residues are evident, as also confirmed uponthe weighing of the scaffolds before and after theleaching process. The interconnectivity of the poresof the scaffolds was tested by slowly pipetting downpropidium iodide/Tween20 solution from the top,allowing sufficient time for the solution to penetratethe entire thickness of the scaffold (following amodified version of the procedure of Widmeret al.30). This dye penetration test indicated that thepores of the scaffolds with 70% or higher percentageporosity (i.e., the eight types of scaffolds designatedas PCL4-9, PCL-TCP1-2 in Table I) were adequatelyinterconnected.

Using a bimodal size distribution of porogen par-ticles increased the porosity and interconnectivity for

TABLE IIScaffold Designations and Their Porosity and BulkCrystallinity Values at 695% Confidence Intervals

Scaffold Designation Porosity (%) Bulk Crystallinity (%)

PCL1 60 6 5 43 6 1PCL2 66 6 1 43 6 1PCL3 70 6 2 42 6 1PCL4 75 6 2 45 6 1PCL5 78 6 3 40 6 3PCL6 75 6 1 40 6 5PCL7 75 6 2 40 6 5PCL8 75 6 1 46 6 1PCL9 73 6 4 46 6 3

PCL_TCP1 72 6 1 44 6 1PCL_TCP2 71 6 1 41 6 5

Figure 2. SEM micrographs (a) and b-TCP nanoparticle concentration distribution (b) of PCL-TCP2 type scaffolds.



the same PCL/DCM concentration [PCL5 shown inFigs. 4(a,b)]. A bimodal particle size distributionincreases the maximum packing fraction of solidparticles in comparison to that of unimodal par-ticles.31,32 Here a weight ratio of 20:80 (small par-ticles over the large particles) was utilized to gener-ate a relatively high packing fraction for the porogenparticle diameter ratio of 25. Figures 4(a,b) (for scaf-fold type PCL5) further indicate that there are pas-sages with typical dimensions of 100 lm, whichshould be suitable for the migration of the osteoblastcells. For the scaffolds of type PCL9, there is a signif-icant variability in the pore sizes. In such structuresthe small pore sizes (typically 5–20 lm) would pre-vent cell migration. However, their interconnectednature [Fig. 4(h)] should still allow the permeationof body fluids.

Figures 4(c,f) show the typical surface topogra-phies of pore surfaces of PCL4 and PCL5 type scaf-

folds. The walls appear to have preferentially-ori-ented and groove-like textures [Figs. 4(c,f)]. Thegroove formation was found to be unaffected bythe incorporation of TCP nanoparticles [compareFigs. 4(c,f) with Fig. 2(a)]. The development ofsuch grooves can be an important structuring effectsince it is known that the cells can align in thegroove direction during their proliferation.33,34 Thegeneration of smaller pore sizes at constant poros-ity resulted in the increase of the surface area andcreated a more fibrous structure with smootherand thinner pore walls as shown in Figures 4(c,f,and i).

Figure 5 shows the spatial distributions of b-TCPnanoparticles within the PCL matrix for scaffolds oftype PCL-TCP2. SEM micrographs indicate that theb-TCP nanoparticles exhibit a broad particle size dis-tribution, that is, 20 nm to 5 l with the notedbreadth of the size distribution consistent with the

Figure 3. SEM micrographs of cryo-fractured PCL7 and PCL8 type scaffolds.



Figure 4. Pore morphology and surface topography of PCL5 type scaffold (a-c), PCL4 type scaffold (d-f) and PCL9 typescaffold (g-i).

Figure 5. SEM micrographs of b-TCP incorporated scaffolds of type PCL-TCP2.

initial size distribution of the b-TCP powder prior toextrusion.

Compressive properties

Compressive properties of the scaffold specimensdecreased with increasing porosity (Fig. 6), similar tothe findings of the earlier studies.35,36 Increasing theporosity from 60 (PCL1) to 75% (PCL4) resulted indecreases of the elastic modulus (slope of the nomi-nal compressive stress versus compressive strain in

the linear region), E, from 363 kPa to 143 kPa andcompressive yield strength, r, from 1214 kPa to 421kPa. The insets in Figure 6 suggest that the wallthicknesses of PCL4 scaffolds are smaller than thoseof PCL1 scaffolds. Decreased quantity of polymerper scaffold volume and a decrease of the extent ofinterconnections should lead to the decrease of com-pressive properties with increasing porosity.

Figure 7 shows that the effects of pore size distri-butions on compressive properties are relativelyminor at constant porosity for the pore size rangesused in the four types of scaffolds of Figure 7, thatis, PCL5-PCL8. Sarazin et al., tested PLLA andPLLA/PCL blend scaffolds with 50% and 60% po-rosity and reported that for the same porosity, theincrease of the pore diameter results in higher com-pressive modulus and strength.37 On the other hand,Quinn et al.38 studied the mechanical properties ofPCL scaffolds, with pore structures that were similarto those of the scaffolds of Sarazin et al.37 but no sig-nificant effects of pore size were detected (consistentwith Fig. 7).

Although the results given in Figure 7 suggestthat changing pore size does not significantly affectthe compressive properties, the results provided inFigure 8 indicate that it is possible to modestly alterthe compressive properties by changing the TCPnanoparticle concentration (increasing compressive

Figure 6. Compressive stress at yield, r, and compressiveelastic modulus, E, values of PCL1-PCL4 scaffolds. Insets:SEM micrographs of PCL1 scaffold at 60% porosity (a)SEM micrographs of PCL4 scaffold at 75% porosity (b).Scale bar represents 200 l.

Figure 7. Compressive stress at yield, r, and compressiveelastic modulus, E, values and SEM micrographs of PCL5,PCL6, PCL7, and PCL8 scaffolds.

Figure 8. Compressive stress at yield, r, and compressiveelastic modulus, E, values of PCL-TCP1 and PCL-TCP2scaffolds. The insets show the corresponding SEM micro-graphs.



properties with increasing TCP concentration). InFigure 8 the compressive properties of a two-layeredscaffold samples of PCL-TCP2 (which contain 48 wt %b-TCP and a mean pore size of 20 lm at the outerlayer and contain 3 wt % b-TCP and a mean poresize of 250 lm at the inner layer) are compared withthose of PCL-TCP1 scaffold samples (which containuniformly distributed TCP nanoparticles at a concen-tration of 3 wt % and a uniform pore size of250 lm). The improvement of the compressive prop-erties upon the incorporation of higher concentra-tions of b-TCP nanoparticles was also observed inother studies at TCP concentrations, which were<50% by wt.39–41

Cell scaffold interactions

Insets of Figures 9(a,b) show the typical opticalmicroscopy and SEM images of hFOB cells after 8days of culture on PCL4 type scaffolds. Both imagesshow that the cells are well spread across the surfaceof the scaffold. SEM micrograph of Figure 9(b)shows the filopodia of an osteoblast attached to thepore walls of the scaffold. Other studies, which werecarried out on 3D porous surfaces, reported the for-mation of a flat cell layer after 24 h.24,42 Coombeset al., reported enhanced hFOB cell attachment andgrowth on microporous PCL surfaces compared tosmooth PCL films.42 This was explained on the basisof the competitive and selective adsorption of celladhesion molecules, such as fibronectin, due toincreased surface area of microporous surfaces and

the positive effect of surface topography on mechani-cal stresses experienced by the cytoskeleton.43,44

Ideally, the height of the protrusions should besmall and the spacing between the protrusionsshould be large enough to enable the clustering ofintegrins, which is a requirement for the formationof mature and stable focal adhesions.45 The typicalsurface topographies of our scaffold surfaces (Fig. 4)indicate that the scale of protrusions on the surfaceis in the 500–1000 nm range. Such grooves shouldnot prevent cell cytoplasm to expand well in alldirections and is expected to trigger the expressionof typical bone differentiation markers.46

The cell viability and proliferation rates were fur-ther investigated by measuring the metabolic activityof the hFOB cells (MTS assay) as a function of timeat 378C. The results for scaffold type PCL4 are givenin Figure 9. It is clear that the metabolic activity ofhFOB cells increased over time, indicating that thehFOB cells were viable and proliferating during the8-days duration of the study. The proliferation ratesobserved in this study are in line with those carriedout earlier with the hFOB 1.19 cell line at 378C.24,26

Alizarin Red Ca2þ quantification assay results aregiven in Figure 10 and indicate that Ca2þ depositionincreased upon 8 days of culturing. Insets of Figure10 show the Ca2þ deposits by the hFOB cells on scaf-fold surfaces. Botchwey et. al. and Yu et. al. also

Figure 9. Optical density, OD, obtained from MTS assay,versus culture time of PCL4 type scaffolds. Insets: (a) opti-cal microscopy upon staining with methylene blue and (b)SEM micrograph, both collected upon 8 days of incuba-tion. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]

Figure 10. Optical density, OD, obtained from Alizarinred assay for Ca2þ deposition. Inset shows the stereo opti-cal microscopy images after ALZ staining. Orange redcolor represents mineralized extracellular matrix forma-tion. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]



reported early mineralized matrix synthesis byosteoblast-like cells after 7 days.21,29

Figure 11 shows the confocal images of hFOB cellnuclei stained with propidium iodide after 7 days inculture and fixation. Confocal microscopy imagescollected from different radial and axial locations ofthe scaffold indicated that the hFOB cells migratedtoward the scaffold center and proliferated. ThehFOB cells achieved a penetration distance of about2500 lm from the seeding surface in 7 days. Asnoted the annular scaffolds developed here for cellpenetration studies are relatively large and have anouter diameter of 8 mm and thickness of 5 mm. Themajority of previous studies with biodegradablepolymer scaffolds were carried out with thinnermembranes, which were usually less then 3 mm inthickness.47–53 This can be an additional advantageof the TSESW technology for the fabrication of scaf-folds suitable for the repair of relatively large tissuedefects.

CONCLUSIONS

Annular scaffolds, which were functionally gradedin terms of pore size and composition, were fabri-cated from PCL incorporated with nanoparticles ofb-TCP, using a TSESW process. The ability to changethe porosity, pore size, and b-TCP nanoparticle con-centration in each layer enabled the investigation ofthe effects of introducing porosity, pore size, andcomposition distributions on compressive properties.Using hFOB cells it was demonstrated that theresulting functionally graded scaffolds are biocom-patible with favorable cell adhesion, penetration,and differentiation rates.

We are grateful to Material Processing and Research Inc.of Hackensack, NJ, for making their MPR 7.5 mm twin-screw-extrusion platform available to us, to ProfessorsArthur Ritter and Antonio Valdevit of Stevens for theirhelp with compressive properties, and to Prof. MatthewLibera of Stevens for his help with SEM usage. We arealso thankful to Dr. David Lagunoff of University of Medi-cine and Dentistry of New Jersey for confocal microscopyand to Professors Susan Szapiel of Rutgers University andArthur Ritter of Stevens for their comments and input.

References

1. Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–926.

2. Mikos AG, Herring SW, Ochareon P, Elisseeff J, Lu HH, Kan-del R, Schoen FJ, Toner M, Mooney D, Atala A, Van DykeME, Kaplan D, Novakovic GV. Engineering complex tissues.Tissue Eng 2006;12:3307–3339.

3. Causa F, Netti PA, Ambrosio L. A multi-functional scaffoldfor tissue regeneration: The need to engineer a tissue ana-logue. Biomaterials 2007;28:5093–5099.

4. Sands RW, Mooney DJ. Polymers to direct cell fate by con-trolling the microenvironment. Curr Opin Biotechnol 2007;18:448–453.

5. Lutolf MP, Hubbell JA. Synthetic biomaterials as instructiveextracellular microenvironments for morphogenesis in tissueengineering. Nat Biotechnol 2005;23:47–55.

6. Leong KF, Chua CK, Sudarmadji N, Yeong WY. Engineeringfunctionally graded tissue engineering scaffolds. J Mech BehavBiomedMater 2008;1:140–152.

7. Pompe W, Worch H, Epple M, Friess W, Gelinsky M, Greil P,Hempel U, Scharnweber D, Schulte K. Functionally gradedmaterials for biomedical applications. Mater Sci Eng A 2003;362:40–60.

8. Xu HH, Burguera EF, Carey LE. Strong, macroporous, and insitu-setting calcium phosphate cement-layered structures.Biomaterials 2007;28:3786–3796.

9. Lawrence BJ, Maase EL, Lin HK, Madihally SV. Multilayercomposite scaffolds with mechanical properties similar tosmall intestinal submucosa. J Biomed Mater Res A 2009;88:634–643.

Figure 11. Confocal laser microscopy of hFOB cells stained with propidium iodide upon 7 days of incubation. Red colorshows the cell nucleus. (a) Penetration depth from cells seeding surface is 50 l (a) and 2.5 mm (b, c, and d). Locations areindicated. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]



10. Wan Y, Feng G, Shen FH, Laurencin CT, Li X. Biphasic scaf-fold for annulus fibrosus tissue regeneration. Biomaterials2008;29:643–652.

11. Ryu W, Min SW, Hammerick KE, Vyakarnam M, Greco RS,Prinz FB, Fasching RJ. The construction of three-dimensionalmicro-fluidic scaffolds of biodegradable polymers by solventvapor based bonding of micro-molded layers. Biomaterials2007;28:1174–1184.

12. Thomas V, Zhang X, Catledge SA, Vohra YK. Functionallygraded electrospun scaffolds with tunable mechanical proper-ties for vascular tissue regeneration. Biomed Mater. 2007;2:224–232.

13. Oh SH, Park IK, Kim JM, Lee JH. In vitro and in vivo char-acteristics of PCL scaffolds with pore size gradient fabri-cated by a centrifugation method. Biomaterials 2007;28:1664–1671.

14. Chen RR, Silva EA, Yuen WW, Mooney DJ. Spatio-temporalVEGF and PDGF delivery patterns blood vessel formationand maturation. Pharm Res 2007;24:258–264.

15. Sherwood JK, Riley SL, Palazzolo R, Brown SC, MonkhouseDC, Coates M, Griffith LG, Landeen LK, Ratcliffe A. A three-dimensional osteochondral composite scaffold for articularcartilage repair. Biomaterials 2002;23:4739–4751.

16. Shao XX, Hutmacher DW, Ho ST, Goh JC, Lee EH. Evalua-tion of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits. Biomaterials 2006;27:1071–1080.

17. Kalita SJ, Bose S, Hosick HL, Bandyopadhyay A. Develop-ment of controlled porosity polymer-ceramic composite scaf-folds via fused deposition modeling. Mater Sci Eng C 2003;23:611–620.

18. Erisken C, Kalyon DM, Wang H. A hybrid twin screw extru-sion/electrospinning method to process nanoparticle-incorpo-rated electrospun nanofibers. Nanotechnology 2008;19:165302.

19. Erisken C, Kalyon DM, Wang H. Functionally graded electro-spun polycaprolactone and b-tricalcium phosphate nanocom-posites for tissue engineering applications. Biomaterials 2008;29:4065–4073.

20. Wang J, Cheung MK, Mi Y. Miscibility and morphology incrystalline/amorphous blends of poly(caprolactone)/poly(4-vinylphenol) as studied by DSC. FTIR and 13C solid stateNMR. Polymer 2002;43:1357–1364.

21. Yu X, Botchwey EA, Levine EM, Pollack SR, Laurencin CT.Bioreactor-based bone tissue engineering: The influence ofdynamic flow on osteoblast phenotypic expression and ma-trix mineralization. PNAS 2004;101:11203–11208.

22. Popat KC, Leary Swan EE, Mukhatyar V, Chatvanichkul KI,Mor GK, Grimes CA, Desai TA. Influence of nanoporous alu-mina membranes on long-term osteoblast response. Biomate-rials 2005;26:4516–4522.

23. Ignatius A, Blessing H, Liedert A, Schmidt C, Neidlinger-Wilke C, Kaspar D, Friemert B, Claes L. Tissue engineeringof bone: effects of mechanical strain on osteoblastic cells intype I collagen matrices. Biomaterials 2005;26:311–318.

24. Jones JR, Tsigkou O, Coates EE, Stevens MM, Polak JM,Hench LL. Extracellular matrix formation and mineralizationon a phosphate-free porous bioactive glass scaffold using pri-mary human osteoblast (HOB) cells. Biomaterials 2007;28:1653–1663.

25. Skelton KL, Glenn JV, Clarke SA, Georgiou G, Valappil SP,Knowles JC, Nazhat SN, Jordan GR. Effect of ternary phos-phate-based glass compositions on osteoblast and osteoblast-like proliferation, differentiation and death in vitro. Acta Bio-mater 2007;3:563–672.

26. Cuddihy MJ, Kotov NA. Poly(lactic-co-glycolic acid) bonescaffolds with inverted colloidal crystal geometry. Tissue EngPart A 2008;14:1639–1649.

27. Subramaniam M, Jalal SM, Rickard DJ, Harris SA,Bolander ME, Spelsberg TC. Further characterization ofhuman fetal osteoblastic hFOB 1.19 and hFOB/ER alphacells: bone formation in vivo and karyotype analysis usingmulticolor fluorescent in situ hybridization. J Cell Biochem2002;87:9–15.

28. Montjovent MO, Burri N, Mark S, Federici E, Scaletta C,Zambelli PY, Hohlfeld P, Leyvraz PF, Applegate LL, PiolettiDP. Fetal bone cells for tissue engineering. Bone. 2004;35:1323–1333.

29. Botchwey EA, Pollack SR, Levine EM, Laurencin CT. Bonetissue engineering in a rotating bioreactor using a micro-carrier matrix system. J Biomed Mater Res 2001;55:242–253.

30. Widmer MS, Gupta PK, Lu L, Meszlenyi RK, Evans GR,Brandt K, Savel T, Gurlek A, Patrick CW Jr, Mikos AG. Man-ufacture of porous biodegradable polymer conduits by anextrusion process for guided tissue regeneration. Biomaterials1998;19:1945–1955.

31. Fiske T, Railkar S, Kalyon DM. Effects of segregation on thepacking of spherical and non-spherical particles. PowderTechnol 1994;81:57–64.

32. Ouchiyama N, Tanaka T. Porosity estimation for randompackings of spherical particles. Ind Eng Chem Fundam1988;23:490–493.

33. Matsuzaka K, Yoshinari M, Shimono M, Inoue T. Effects ofmultigrooved surfaces on osteoblast-like cells in vitro: Scan-ning electron microscopic observation and mRNA expressionof osteopontin and osteocalcin. J Biomed Mater Res A 2004;68:227–234.

34. Degirmenbasi N, Ozkan S, Kalyon DM, Yu X. Surface pat-terning of poly(L-lactide) upon melt processing: in vitro cul-turing of fibroblasts and osteoblasts on surfaces ranging fromhighly crystalline with spherulitic protrusions to amorphouswith nanoscale indentations. J Biomed Mater Res A 2009;88:94–104.

35. Karageorgiou V, Kaplan D. Porosity of 3D biomaterialscaffolds and osteogenesis. Biomaterials 2005;26:5474–5491.

36. Thomson RC, Yaszemski MJ, Powers JM, Mikos AG. Fabrica-tion of biodegradable polymer scaffolds to engineer trabecu-lar bone. J Biomat Sci Polym Ed 1996;7:23–38.

37. Sarazin P, Roy X, Favis BD. Controlled preparation and prop-erties of porous poly(L-lactide) obtained from a co-continuousblend of two biodegradable polymers. Biomaterials 2004;25:5965–5978.

38. Quinn TP, Oreskovic TL, Landis FA, Washburn NR. Materialmodel measurements and predictions for a random pore poly(epsilon-caprolactone) scaffold. J Biomed Mater Res B ApplBiomater 2007;82:205–209.

39. Shor L, Guceri S, Wen X, Gandhi M, Sun W. Fabrication ofthree-dimensional polycaprolactone/hydroxyapatite tissuescaffolds and osteoblast-scaffold interactions in vitro. Bioma-terials 2007;28:5291–5297.

40. Wei G, Ma PX. Structure and properties of nano-hydroxyapa-tite/polymer composite scaffolds for bone tissue engineering.Biomaterials 2004;25:4749–4757.

41. Causa F, Netti PA, Ambrosio L, Ciapetti G, Baldini N, PaganiS, Martini D, Giunti A. Poly-epsilon-caprolactone/hydroxy-apatite composites for bone regeneration: In vitro characteri-zation and human osteoblast response. J Biomed Mater Res A2006;76:151–162.

42. Coombes AG, Rizzi SC, Williamson M, Barralet JE, DownesS, Wallace WA. Precipitation casting of polycaprolactone forapplications in tissue engineering and drug delivery. Bioma-terials 2004;25:315–325.

43. Nimni ME. Polypeptide growth factors: targeted delivery sys-tems. Biomaterials 1997;18:1201–1225.



44. Ingber DE. Mechanical and chemical determinants of tissuedevelopment. In: Lanza RP, Langer R, Vacanti J, editors. Prin-ciples of Tissue Engineering. San Diego: Academic Press;2000. p 101–110.

45. Kunzler TP, Huwiler C, Drobek T, Voros J, Spencer ND. Sys-tematic study of osteoblast response to nanotopography bymeans of nanoparticle-density gradients. Biomaterials 2007;28:5000–5006.

46. Liao H, Andersson AS, Sutherland D, Petronis S, Kasemo B,Thomsen P. Response of rat osteoblast-like cells to micro-structured model surfaces in vitro. Biomaterials 2003;24:649–654.

47. Lu JX, Flautre B, Anselme K, et al. Study of porous intercon-nections of bioceramic on cellular rehabitation in vitro and invivo. In: Sedel L, Rey C, editors.Bioceramics 10. Paris: Perga-mon, Elsevier Science, 1997. p 583–586.

48. Ishaug-Riley SL, Crane-Kruger GM, Yaszemski MJ, MikosAG. Three-dimensional culture of rat calvarial osteoblasts in

porous biodegradable polymers. Biomaterials 1998;19:1405–1412.

49. Heinrich L, Freyria AM, Melin M, Tourneur Y, Maksoud R,Bernengo JC, Hartmann DJ. Confocal laser scanning micros-copy using dialkylcarbocyanine dyes for cell tracing in hardand soft biomaterials. J Biomed Mater Res B Appl Biomater2007;81:153–161.

50. Degirmenbasi N, Kalyon DM, Birinci E. Biocomposites ofnanohydroxyapatite with collagen and poly(vinyl alcohol).Colloids and Surfaces B Biointerfaces. 2006;48:42–49.

51. Yoshimoto H, Shin YM, Terai H, Vacanti JP. A biodegradablenanofiber scaffold by electrospinning and its potential forbone tissue engineering. Biomaterials 2003;24:2077–2082.

52. Wei G, Jin Q, Giannobile WV, Ma PX. The enhancement ofosteogenesis by nano-fibrous scaffolds incorporating rhBMP-7nanospheres. Biomaterials 2007;28:2087–2096.

53. Malda J, Klein TJ, Upton Z. The roles of hypoxia in the invitro engineering of tissues. Tissue Eng 2007;13:2153–2162.



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