Three-Dimensional Hierarchical Composite ... - ACS Publications

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Biomacromolecules 2011, 12, 502–510

Three-Dimensional Hierarchical Composite Scaffolds Consisting of Polycaprolactone, β-Tricalcium Phosphate, and Collagen Nanofibers: Fabrication, Physical Properties, and In Vitro Cell Activity for Bone Tissue Regeneration MyungGu Yeo,† Hyeongjin Lee,† and GeunHyung Kim*,†,‡ Bio/Nanofluidics Lab, Department of Mechanical Engineering, Chosun University, Gwangju 501-759, Korea, and Department of Dental Life Science, College of Dentistry, Chosun University, Gwangju 501-759, Korea Received November 2, 2010; Revised Manuscript Received December 1, 2010

β-Tricalcium phosphate (β-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to β-TCP’s inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/β-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the β-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter ) 160 nm) were well layered between the composite struts. By accommodating the β-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/β-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/β-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and β-TCP particles in the scaffold provide good synergistic effects for cell activity.

1. Introduction The use of biomedical scaffolds has been widely explored as a method to replace damaged tissues and organs. The required properties of the biomaterials used in these scaffolds include biodegradability, biocompatibility, and a three-dimensional (3D) structure. The 3D structure should be fully porous with an interconnected-pore structure to allow vascularization, cell migration, and nutrient transfer from the surface to the internal area of the structure.1-3 In addition, the 3D structure should have the appropriate biological and physical properties, including surface topography and mechanical sustainability.3,4 At present, although studies have sought to establish the ideal scaffold structure for tissue regeneration, an optimized 3D structure has not been determined.4-6 To achieve an appropriate scaffold for bone tissue regeneration, Bioceramics including hydroxyapatite, bioactive glasses, and calcium phosphate ceramics have been widely researched due to their prominent biological properties (osteoconduction and osteoinductive properties for the case of 3D pore structures).7,8 Specifically, a β-tricalcium phosphate (β-TCP) polymorph shows higher bioresorption and osteoconductivity in the physiological environment than the R-form of TCP.9-11 However, Bioceramics have certain limitations including inherent brittleness, which makes applying various directions of loading forces * To whom correspondence should be addressed. Tel.: +82-62-230-7180. Fax: +82-62-236-3634. E-mail: [email protected]. † Department of Mechanical Engineering. ‡ Department of Dental Life Science.

difficult, and low controllability of degradation due to osteoclastic activity.8 To enhance mechanical integrity and the controllability of degradation, bioceramic materials have been sintered at high temperatures, and in most cases, mixtures (blend/ composite) with various polymers [polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA), and polyglycolic acid (PGA)] have been introduced.12,13 Of these materials, PCL is one of the most attractive synthetic polymers. PCL has been fabricated with β-TCP, resulting in a composite that can be used for bone tissue engineering scaffolds.12,14-16 In general, the 3D shape of bone tissue substitute (scaffold) and its mechanical stability have been a major issue in bone tissue regeneration. To generate 3D structured scaffolds, solid free-form fabrication including precision extrusion deposition (PED), bioplotting, stereolithography, and selective laser sintering have been applied.17,18 Recently, Sun et al. fabricated a 3D strut-structured PCL/hydroxyapatite (HA) scaffold for bone tissue regeneration using the PED method.19 They found that layered 3D PCL/HA composite scaffolds had a higher expression of alkaline phosphatase activity (ALP) and mineralization as compared to a pure PCL scaffold. In addition, Arafat et al. fabricated a new composite that consisted of a 3D structured PCL/TCP coated with carbonated hydroxyapatite-gelatin.15 They found that the porcine bone marrow stromal cells cultured on layered 3D PCL/HA composite scaffold coated with gelatin showed a 2.3 times higher proliferation rate as compared to cells on to a PCL/TCP scaffolds after 10 days of cell culture. They hypothesized that the gelatin served as functionalized cell

10.1021/bm1013052  2011 American Chemical Society Published on Web 12/28/2010

Three-Dimensional Hierarchical Composite Scaffolds

Biomacromolecules, Vol. 12, No. 2, 2011

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Figure 1. Schematic of fabrication processes for producing composite scaffolds and hierarchical scaffolds that consist of PCL, β-TCP particles, and electrospun collagen nanofibers.

proliferation sites. However, although cell growth on the scaffold strut had increased dramatically after 31 days of cell culture, cell compactness between pores of the scaffold was still low. Previously, our group attempted to address cell compactness by using hierarchical scaffolds with PCL struts and electrospun PCL nanofibers for cartilage regeneration.20 We found that cell proliferation improved greatly and cell compactness was dramatically enhanced over a short cell culturing time. However, cell attachment and proliferation mainly occurred in the electrospun fibers within the hierarchical scaffolds. The goal of the present research was to resolve this problem. To address this difficulty, we fabricated a hierarchical composite scaffold that consisted of PCL/β-TCP struts and electrospun collagen nanofibers, which is one of the main organic components of bone.5 To determine the effects of β-TCP in the PCL struts and electrospun collagen fibers, we fabricated composite scaffolds with three different compositions (0, 20, 40 wt %) of β-TCP in the layered PCL struts with or without electrospun collagen fibers. The fabricated hierarchical scaffolds were assessed for surface morphology, TCP composition, and tensile modulus. We then compared these result with those obtained using composite scaffolds without electrospun collagen nanofibers. In addition, water-absorbability, hydrophilic properties, and biological capabilities of these scaffolds were also evaluated by culturing osteoblast-like cells (MG63).

2. Experimental Section Materials. PCL (Mw, 80000; melting point, 60 °C) was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.), and bioceramic (β-TCP) powders were purchased from Fluka (St. Louis, MO, U.S.A.). The measured particle size distributions of β-TCP were between 100 nm and 12 µm. Type-I collagen (Matrixen-PSP; Bioland, Cheonan City, South Korea) from porcine tendon was used for the electrospinning process and it was deposited on the surface of the PCL/β-TCP struts. To fabricate electrospun collagen fibers, which were layered between the composite struts, we dissolved 4 wt % collagen in a 1,1,1,3,3,3hexafluoro-2-propanol solvent and injected the solution through a nozzle (21 G) using 20 mL glass syringes. To cross-link the electrospun collagen nanofibers, a 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Mw 191.7; Sigma-Aldrich) solution was used. Scaffold Fabrication. As shown in Figure 1, to fabricate 3D hierarchical composite scaffolds, we used two combined processes: a melt plotting connected to a three-axis robot system to draw the mixture of

PCL and β-TCP particles and an electrospinning system to generate collagen nanofibers. The mixture of PCL powders and β-TCP powders was injected into a heating cylindrical cartridge at 115 °C. The melted PCL/β-TCP was extruded through a heated 250-µm needle tip with constant pressure. The extruded strands were reground in a freezer mill to obtain a uniform distribution of β-TCP in PCL resin during the extrusion process. The reground mixture was again transferred to the cylindrical cartridge of the machine, and the perpendicular composite struts were plotted on a plotting stage. The temperature of the processing and ambient temperature of the plotting system were fixed at 115 and 20 °C, respectively. The mixture was drawn by controlling the pneumatic pressure (680 ( 47 kPa) and plotted with a 250 µm needle tip. First, PCL or PCL/β-TCP were transferred to the cylindrical cartridge of the plotter and the perpendicular PCL or PCL/β-TCP struts were plotted on a plotting stage to complete one layer. Second, the upper stage connected to the electrospinning apparatus was moved automatically to the layered struts, and then collagen nanofibers were electrospun on top and this process is repeated several times to fabricate a 3D scaffold. Electric fields used in this process were 0.17-0.22 kV mm-1, the electrospinning deposition time on the struts was 1 min, and the flow rate of the collagen solution was fixed at 0.2 mL h-1 using a syringe pump (KDS 230; KD Scientific, Holliston, MA, U.S.A.). A power supply (SHV300RD-50K; Convertech, Seoul, South Korea) was used to provide a high electrical field. To cross-link the collagen fibers in the hierarchical scaffold, it was immersed in 50 mM EDC solution in 95% ethanol for 24 h at room temperature. The fabricated scaffolds were washed several times with a phosphate-buffered saline (PBS) solution and finally with distilled water to remove the unreacted EDC solution. Scaffold Characterization. The structural morphology of the scaffolds was observed under an optical microscope (BX FM-32; Olympus, Tokyo, Japan) connected to a digital camera and a scanning electron microscope (SEM; Sirion, Hillsboro, OR, U.S.A.). To evaluate quantitative amounts of β-TCP in the PCL struts, wideangle X-ray diffractometry (WAXD) measurements were performed with a diffractometer (D/Max-2500, 18 kW; Rigaku, Tokyo, Japan) at 40 kV and 200 mA using sealed-tube Cu KR (1.542 Å) radiation collimated by a graphite monochromator. A Fourier-transform infrared (FTIR) spectrometer (model 6700; Nicolet, West Point, PA, U.S.A.) was used for measuring the crosslinking of electrospun collagen nanofibers. IR spectra represent the average of 30 scans between 400 and 4000 cm-1 at a resolution of 8 cm-1. Water absorption was calculated by weighing the scaffolds before and after soaking in distilled water for 2 h, in accordance with the methods described by of Li et al.21 The percent increase in water absorption was calculated as (%) ) (W2h - Wo)/Wo × 100, where W2h

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is the weight of the scaffold after 2 h and Wo is the original weight of the scaffold at time zero. The water contact angle (WCA) of the scaffolds was measured using a contact angle analyzer (Phoenix 300; SEO Surface and Electro-Optics, Suwon City, South Korea). The contact angles for five independent scaffolds were averaged and are presented as the mean ( standard deviation (SD) of five different specimens. A water droplet of 10 µL was placed on the surface of the fabricated scaffolds and the contact angle measured under the atmospheric temperature of 23.2 °C and humidity of 37%. Mechanical properties of the scaffolds were evaluated in a dry state using the tensile mode. The scaffolds were cut into small strips (5 × 15 × 3 mm). For each scaffold, five samples were obtained from different sites. The size of the specimens was considered as a rectangular shape and was measured using a digital caliper micrometer (Ultracal III; Sylvac, Bern, Switzerland). To obtain the size, three different parts of the specimen were measured and averaged. The tensile test was characterized using a universal tensile machine (Top-tech 2000; Chemilab, Suwon, South Korea). The stress-strain curves of the scaffolds were recorded at a stretching speed of 2 mm s-1. The apparent porosity of the scaffolds was obtained by the following equations.22

porosity(%) ) 1 -

apparent density of scaffold bulk density of scaffold

apparent density ) weight of scaffold volume of scaffold assumed as a rectangular shape The density of the composites was obtained using the rule of mixture. The bulk densities of PCL and β-TCP were 1.135 and 3.14 g cm-3, respectively. The weight percent of β-TCP in the composites was changed to a volume fraction (φc). This was calculated using the simple equation22

φc )

ωp ωp(1 - λ) + λ

where ωp is the weight fraction of β-TCP, λ ) Fp/Fm, and Fp and Fm are the density of β-TCP and PCL, respectively. The density of the composite consisting of PCL and 20 wt % (8 vol %) β-TCP was 1.30 g cm-3 and the density of the composite consisting of PCL and 40 wt % (20 vol %) β-TCP was 1.54 g cm-3. The scaffolds were weighed with a precise balance (AD-4 autobalance; Perkin-Elmer, Waltham, MA, U.S.A.). Pore size was assumed to be the distance between PCL struts and was determined by optical microscopy and SEM. Cell Culturing. Scaffolds for use with cell cultures, 5 × 5 × 3 mm3, were sterilized with 70% EtOH and UV light, and placed in culture medium overnight. MG63 cells (ATCC, Manassas, VA, U.S.A.) were used to observe cellular behavior in the scaffolds. MG63 cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, U.S.A.) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Hyclone). The cells were maintained up to passage 7 and collected by trypsin-EDTA treatment. The cells were then seeded onto the scaffolds at a density of 5 × 104 cells per sample and incubated in an atmosphere of 5% CO2 at 37 °C. To assess the morphology of cells on the scaffolds, the cells were examined by SEM after 7 days. The cell/scaffold constructs were fixed in 2.5% glutaraldehyde and dehydrated through a graded ethanol series. Dried scaffolds were coated with gold and examined under SEM. Cell growth was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (Cell Proliferation Kit I; Boehringer Mannheim, Mannheim, Germany). This assay is based on the cleavage of the yellow tetrazolium salt MTT by mitochondrial dehydrogenases

Yeo et al. in viable cells to produce purple formazan crystals. Cells on the scaffold were incubated with 0.5 mg mL-1 MTT for 4 h at 37 °C and the absorbance at 570 nm was measured using a microplate reader (EL800, Bio-Tek Instruments, Winooski, VT, U.S.A.). Statistical Analysis. All data presented are expressed as the mean ( SD. Statistical analyses consisted of single-factor analyses of variance (ANOVA). The significance level was set at p < 0.05.

3. Results and Discussion Fabrication of Hierarchical Composite Scaffolds. The pore structure of scaffolds used for bone tissue regeneration is an extremely important factor because the porosity and pore interconnectivity critically influence neovascularization and transportation of nutrients and oxygen from the surface to the rest of the scaffold.23 In this study, the PCL/β-TCP composite and hierarchical composite scaffolds were fabricated using bioplotting and an electrospinning system. When using the bioplotting system with a melted synthetic polymer, the strut diameter is a very important processing parameter because the fabricated diameter can influence pore size (distance between the struts) and porosity. Therefore, the controllability of strut diameter is a key processing condition. When the melted polymer is extruded through the nozzle, the extruded strut can be highly swelled at the nozzle tip compared to the nozzle diameter, and the swelled strut diameter is critically dependent on both the volume percent of β-TCP in the PCL composite and nozzle speed.24,25 This phenomenon can be explained by the macroscopic rheology of the melted polymer composite. The die-swell of a polymer can result from elastic recovery of elongational and shear deformation in the microsized nozzle as the melted polymer leaves the nozzle.24,25 Main effects include the volume fraction of particles in the polymer resin, temperature, extrusion rate, and the channel’s length over diameter.26 In our PCL/β-TCP system, as the volume fraction of the β-TCP increased, the dissipation of stored elastic deformation energy and blockage of the elastic recovery of deformed PCL also increased, resulting in a reduction of the die-swell ratio (ds/d; ds: the diameter of strut, d: a nozzle diameter) of the composite system. Figure 2a shows the strut diameters of pure PCL and the P/T composite (PCL/β-TCP) for various nozzle moving speeds and weight percents of β-TCP (nozzle diameter was 250 µm). The pure PCL showed a high die-swelling value, while the composites showed relatively lower values. Additionally, in the 20 and 40 wt % β-TCP in the PCL composites, the diameters of the composite struts were similar. By using a simple process diagram to obtain similar strut sizes for pure PCL and the composites, we could appropriately change the moving speed of the nozzle. To fabricate uniform strut size for pure PCL and various composites, various moving speeds of 0.32, 0.25, and 0.18 mm s-1 were applied for PCL, PCL/TCP (20 wt %), and PCL/TCP (40 wt %), respectively. To observe the distribution of β-TCP particles in the composite strut, a typical SEM micrograph of the strut cross section consisted of PCL and 40 wt % β-TCP, as shown in Figure 2b. The concentration versus radial distance distribution of Ca in the strut as determined by EDX is presented in Figure 2c. In the graph, Ca (%) was defined as [(an EDX intensity of Ca for one area) × 100]/[total EDX intensity of Ca for three areas]. As shown in the results, β-TCP particles within the composite strut were well embedded. Surface and Cross-Sectional Morphologies of Fabricated Scaffolds. Figure 3a,b shows optical and SEM images of pure PCL, PCL/β-TCP (40 wt %), and hierarchical composite scaffolds. The fabricated pore size of the scaffolds was fixed



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diameter was measured as 160 ( 80 nm [Figure 3d], and the average pore diameter (D), which consisted of collagen fibers, was determined using D ) [(4 × A)/π]1/2 assuming circular pores, where A is the pore area. The average pore size of the collagen fibers was 3.37 ( 1.3 µm [Figure 3e].

Figure 2. (a) Diameter differences of struts for various weight percents of β-TCP and nozzle speeds. (b) Typical SEM image of a cross section of PCL/β-TCP (40 wt %). (c) Ca2+ content (%) at various positions in a strut (PCL/TCP-40 wt %). NS indicates nonsignificance.

from 250 to 300 µm because the optimal pore size of scaffolds for bone regeneration is suggested to be between 200 and 400 µm for osteoconduction.27 The diameter of the composite strut was fixed at 300 ( 20 µm and interconnectivity between pores in the scaffolds was 100%. The magnified image in Figure 3c clearly indicates that the electrospun collagen nanofibers were located between the layers of plotted PCL/β-TCP struts. Using the SEM image from Figure 3c, average collagen nanofiber

SEM micrographs of surface and cross-sectional views of PCL, composites, and hierarchical composite scaffolds are presented in Figure 4. As shown in Figure 4a-c, the PCL strut was well fabricated. Figure 4d-f show that the composite scaffold fabricated with PCL and 20 wt % (8 vol%) β-TCP. The composite of PCL and 40 wt % (20 vol%) β-TCP is shown in Figure 4g-i. Based on these images, the surface roughness on the strut was higher relative to the composite (20 wt % β-TCP in PCL). In the crosssectional image, the mixed β-TCP particles were well embedded within the PCL struts. In addition, as increasing the volume fraction of β-TCP particles in PCL, some voids or defects on the strut surface were found [Figure 4g,j]. We think that this phenomenon can be because of the clusters of the β-TCP particles, which are not allowing an intimate mixing with the PCL. As shown in the SEM images of Figure 4, β-TCP powders were well embedded, but uniform distribution of β-TCP powders was not acquired in the PCL composite strut due to the low mix-ability of the powders in the PCL strut. However, while pure PCL scaffolds exhibited a smooth surface morphology, the two composite scaffolds appeared with a rough surface morphology, although β-TCP particles in the melted PCL could be moved to the core area of the strut to reduce shear stress during the extrusion process. Figure 4j-l describe the hierarchical composite scaffolds consisting of PCL, 40 wt % β-TCP particles, and electrospun collagen nanofibers. As shown in the images, the collagen nanofibers were well set between the composite struts in the surface and thickness directions. As shown in the SEM images, we could confirm that the β-TCP particles and collagen fibers were well embedded between the PCL struts and that the pore structure of the scaffolds was well controlled.

Figure 3. Optical and scanning electron microscope images of (a) a PCL scaffold fabricated using a normal plotting system (the lower picture shows the side of the scaffold), (b) the composite scaffold (P/T-40%) consisting of PCL and 40 wt % β-TCP, and (c) the hierarchical composite scaffold (P/T-40%/C; the magnified pictures show collagen fibers between the PCL/TCP composite struts). (d) Size distribution of electrospun collagen fibers in the hierarchical scaffold. (e) The pore-size distribution consisted of collagen fibers within the pores of the scaffold.

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Figure 4. SEM images of (a) a PCL scaffold fabricated using the melt-plotting system and (b) surface and (c) cross section of a strut; (d-f) a composite scaffold (PCL and 20 wt % β-TCP); (g-i) a composite scaffold consisting of PCL and 40 wt % β-TCP; (j-l) a hierarchical composite scaffold with collagen nanofibers (the magnified pore shows the collagen nanofibers set between struts).

Figure 5. X-ray diffraction (XRD) patterns of the pure PCL and PCL composites for various weight percents of β-TCP: (a) pure PCL, (b) PCL/TCP (20 wt %), (c) PCL/TCP (40 wt %), and (d) pure TCP.

X-ray diffractometer patterns of pure PCL, TCP, and composites of PCL/β-TCP are described in Figure 5. All composite patterns showed two strong peaks located around 2θ ) 21.4° and 23.8° of PCL, which were associated with the (110) and (200) reflections of a polyethylene-like crystal structure28 with orthorhombic unit cell parameters of a ) 0.748 nm, b ) 0.498 nm, and c ) 1.729 nm,28,29 together with the peaks of the β-TCP particles. The reflection intensities of β-TCP in the composite were qualitatively shown to increase with an increase in the content of the weight percent of β-TCP. To cross-link the collagen fibers in the hierarchical scaffolds of PCL/β-TCP, the scaffold was immersed in a 50 mM EDC solution in 95% ethanol for 24 h at room temperature. Figure 6

shows the FTIR spectra of the electrospun collagen fibers before and after cross-linking. In the figure, the N-H stretching vibration peak is approximately 3324 cm-1 and the amide I (1635 cm-1), amide II (1536 and 1458 cm-1), and amide III bands (1211, 1235, and 1268 cm-1) are also visible. The amide I, II, and III bands of collagen are directly related to the polypeptide conformation. In general, when determining the degree of collagen curing, amide bands have been used due to the formation of ester linkages resulting from carboxyl groups reacting with the amide and hydroxyl groups of collagen.30 As shown in Figure 6b, after cross-linking collagen nanofibers, the amide groups decreased due to the linkage of hydroxyl groups. These IR results suggest that the collagen nanofibers in the scaffold were cross-linked in the EDC solution. Water-Uptake Ability of Fabricated Scaffolds. The wateruptake ability of the scaffold can influence cell proliferation and the structural morphology of the tissue.4 Figure 7 shows the water absorption of various composites with and without electrospun collagen nanofibers. In general, β-TCP can be a water absorption site in various polymer composites, such that water-uptake ability can be increased compared to pure synthetic polymers.31 As shown in Figure 7, as the concentration of β-TCP increased, the water uptake also increased. However, although a high weight percent of β-TCP was added to the PCL resin, the degree of water uptake was still low because the water uptake was only related to the surface of the struts. The low degree of water uptake can be improved by using collagen nanofibers. The collagen nanofibers layered in the composite scaffold acted as good water uptake sites, such that water uptake



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Figure 6. FTIR spectra of collagen nanofibers of the hierarchical composite scaffold before and after cross-linking with EDC solution.

Figure 7. Water absorption of pure PCL and PCL/β-TCP (P/T) composite scaffolds with and without collagen nanofibers. NS indicates nonsignificance.

increased about 80-160% compared to composites without collagen fibers. This result suggests that the layered collagen nanofibers in the composite scaffold might be good waterabsorption sites that can be used to prevent the loss of body fluid and nutrients in in vivo tests. Hydrophilic Properties of Fabricated Scaffolds. Several studies have revealed that hydrophilic properties can influence osteoblast adhesion and proliferation. Several methods including physical patterning32 and chemical modification33 have been used to increase the hydrophilicity of scaffolds. The increased hydrophilic properties of the scaffold can affect osteoblast cell attachment and spreading; human osteoblastic cells preferentially adhere in hydrophilic patterns on nanocrystalline diamond film.32 In addition, blending and composite systems using hydrophilic materials such as cell adhesive protein,34 poly(vinyl alcohol),35 and β-TCP31 have been used to increase the hydrophilic properties of scaffolds. To examine the effects of layered collagen nanofibers on the hydrophilicity of hierarchical composite scaffolds, WCAs were measured and compared to those of pure PCL and various composite scaffolds with and without collagen nanofibers. In general, since collagen is a very hydrophilic material, we predicted that the inserted collagen fibers may play a prominent role in increasing the hydrophilic properties of the hierarchical scaffolds. As shown in Figure 8a-f, the WCA of droplets on various composite scaffolds showed relatively low values compared to that of the pure PCL scaffold. However, as shown in Figure 8g, the hierarchical scaffolds supplemented with collagen nanofibers showed a more meaningful decrease in WCA as compared to the composite scaffolds. In addition, the degree of increased hydrophilicity of the hierarchical scaffold was not correlated with the weight percent of the mixed β-TCP powders.

Tensile Properties. For bone tissue regeneration, the scaffold should not only possess biologically advantageous aspects as a cell carrier, but also adequate mechanical properties that can be maintained until neo-tissues are regenerated in the damaged area and replaced naturally. Collagen has been widely used a biomaterial, but its poor mechanical properties, including low mechanical strength, have limited its application as a scaffold for bone tissue regeneration. In the present study, we attempted to generate a scaffold that has the same biological functions as collagen, but also a mechanically stable structure complemented with PCL and β-TCP. Figure 9 shows a comparison of the tensile modulus between the composite scaffolds with and without collagen fibers at a stretching speed of 2 mm s-1. Tensile tests were conducted using a universal tensile machine at 30 °C. The Young’s modulus of the scaffolds ranged from 6 to 9 MPa. According to the modified Halpin-Tsai equation,36

ECOM )

ζ)

[

5 1 + 2ξφ E + [ 83 1 +1 -0.67ζφ ζφ 8 1 - ξφ ]

(1)

PCL

]

ETCP /EPCL - 1 , ETCP /EPCL + 0.67

ξ)

[

ETCP /EPCL - 1 ETCP /EPCL + 2

]

(2)

where E6 and φ are the Young’s modulus of the composite and the volume fraction of β-TCP, respectively, and E6 and E6 are the Young’s moduli of β-TCP and PCL, respectively. By increasing the volume fraction of the dispersed phase in the composite, the mechanical properties can be improved. Based on eqs 1 and 2, because 6ETCP . 6EPCL (ξ ≈ 1, ζ ≈ 1), the tensile modulus of the composites including 20 and 40 wt % β-TCP in PCL can be calculated as 8.16 and 11.0 MPa, respectively. The calculated values were higher than those of the measured modulus. The difference between the theoretically obtained and measured moduli may be due to the TCP particle’s microstructural homogeneity in the continuous phase and interfacial adhesion between the dispersed phase and continuous phase.37 In addition, the pore structure (porosity) of the scaffold was another factor responsible for the low mechanical properties, as compared to the PCL/β-TCP block. However, we can confirm that the modulus was affected by the volume fraction of β-TCP particles, while the layered collagen nanofibers in the scaffold did not influence the final mechanical properties because only a small amount of collagen nanofibers was inset in the scaffold. Details on the porosity and modulus of the scaffolds are described in Table 1. Statistical analysis using a one-way

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Figure 8. WCA of the PCL, composites (P/T), and hierarchical composite scaffolds (P/T/C). (a-f) Water droplets at two time points (1 s and 5 min) for various scaffolds. (g) Comparisons of the WCAs of the various scaffolds.

Figure 9. Comparison of Young’s modulus between PCL and composite scaffolds (n ) 5). Table 1. Porosity and Tensile Modulus of Various Composites and Hierarchical Scaffolds at a Constant Stretching Speed of 2 mm s-1 scaffold PCL PCL/collagen PCL/TCP (20 PCL/TCP (20 PCL/TCP (40 PCL/TCP (40

wt wt wt wt

%) %)/collagen %) %)/collagen

porosity (%)

Young’s modulus (MPa)

55.8 55.2 55.1 54.7 55.4 54.1

6.87 ( 1.07 6.92 ( 0.86 7.54 ( 0.99 7.48 ( 0.85 8.34 ( 0.77 8.47 ( 0.97

ANOVA showed that no significant difference was observed in the tensile modulus between the composites with and without collagen nanofibers (p > 0.05). In Vitro Tests of MG63 Cells. The interaction between cells and the scaffold should be considered because the scaffolds can be optimally designed to grow cells. The dispersed phase, β-TCP, is very similar to bone minerals with respect to bioactivity. β-TCP can be osteoconductive and also has osteoinductive properties when it is designed with the appropriate

geometry and topography combined with microporosity in which various growth factors and bioactive materials including collagen and bone morphogenetic proteins (BMPs) can be entrapped.7 Figure 10a,h show the SEM images of MG63 cells attached in the PCL, PCL/β-TCP (40 wt %) composite, and hierarchical scaffold (PCL/β-TCP (40 wt %)/collagen nanofibers) with the pore range of 250-300 µm after 7 days of cell culture. In this study, we evaluated the influence of collagen nanofibers layered in the PCL/β-TCP composite scaffold on the morphology and proliferation of MG63 cells. As shown in the SEM images (Figure 10a), on day 7, MG63 had a rounded morphology on the surface of the PCL strut. The cells did not sufficiently proliferate on the surface of the PCL struts and the pores of the scaffold were not filled with cells. However, in the case of the neat PCL scaffold that was inserted with collagen nanofibers, the cells were well adhered (Figure 10b). This indicates that the MG63 cells can easily adhere and proliferate better on the hierarchical scaffold as compared to a pure PCL scaffold. However, in the hierarchical scaffolds that consisted of pure PCL and collagen fibers, the cells only adhered in the collagen fibers, rather than on the surface of the strut (Figure 10b). For the PCL/TCP composite, the β-TCP particles were used to increase cell attachment and proliferation on the struts. The composite PCL/TCP scaffold showed increased cell attachment compared to the pure PCL scaffold (Figure 10c-e), but the cell compactness between the pores was still too low. In addition, low cell proliferation into the interior of plotted PCL/TCP scaffolds was observed, although the pore interconnectivity was 100% and adequate pore size was generated in the scaffold. In the case of the hierarchical composite scaffold, the collagen nanofibers and composite struts that included TCP induced high cell attachment and proliferation, resulting in a uniform cell distribution throughout the struts and pores of the scaffold (Figure 10f-h). This phenomenon is noticeably different from the cell proliferation behavior of the PCL hierarchical scaffold



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Figure 10. SEM micrographs of MG63 cells cultured on (a) a pure PCL scaffold, (b) hierarchical PCL scaffold with collagen fibers, (c-e) PCL/ β-TCP (40 wt %), and (f-h) a hierarchical composite scaffold consisting of PCL/β-TCP (40 wt %) and collagen nanofibers after 7 days of cell culture. (i, j) EDX images for the hierarchical scaffolds without and with TCP.

only supplemented with collagen nanofibers (Figure 10b) and the PCL/TCP composite scaffold without collagen fibers (Figure 10c-e). In addition, from the EDX imaging the amount of

elemental Ca and P was characterized from the cell surfaces of the cell-cultured scaffolds. Figure 10i,j shows the results of day 7 EDX imaging for the scaffold of Figure 10b and the

Figure 11. (a) Cell proliferation as indicated by the MTT assay of MG63 cells seeded on various scaffolds. (b) Comparisons of cell growth rates for various scaffolds. *p < 0.05 indicates a significant difference.

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hierarchical composite scaffold of Figure 10h, respectively. As shown in Figure 10i,j, the Ca was found on the hierarchical composite scaffold, while in the scaffold consisted of pure PCL and collagen nanofibers it was not found. This highly enhanced cell proliferation rate and mineralization of the scaffold might have been a result of calcium and phosphate ions released from TCP that are met with nanofibrous collagen (this process is chemical precipitation). When this occurs, some synergistic effects may be obtained and evoke the dramatic cell proliferation over a short cell culturing time, although we cannot provide evidence for this phenomenon. Cell activity was also consistent with the results of WCA and water-absorption ability. In Figure 11a, MG63 cell proliferation on the PCL, composite [PCL/β-TCP (40 wt %)], and hierarchical scaffolds were measured using the MTT assay. As shown in the figure, cells on all scaffolds proliferated well with time. However, initial cell attachment data indicate that significant differences existed between the PCL and hierarchical scaffolds, but not between PCL and the composite scaffold. This phenomenon can be explained by the previous SEM images, which showed that cells easily attach in the collagen nanofiber layer. After 7 days of culture, the hierarchical composite scaffold showed the highest cell proliferation rates compared to any other scaffold due to the synergistic effects of β-TCP particles and collagen nanofibers. To compare cell proliferation rates in the scaffolds, we defined the rate of cell proliferation as the slope of the optical density (OD) to cell culturing days. The increasing rate was almost a linear relation between the OD value vs culturing days. Model fitting was conducted using commercial software (Origin 7.0; OriginLab, Northampton, MA, U.S.A.). Based on the cell growth rate results (Figure 11b), we could determine that the collagen nanofibers and β-TCP particles on the strut promote initial cell attachment and accelerate cell proliferation during the culturing time. This phenomenon can be observed on the SEM images of Figure 10d-f. When we compared the images in Figure 10b,c with that in Figure 10f, we found that the MG63 cells were completely packed in the pores and covered the surface of the struts. However, at this time, we cannot exactly explain the synergic effects caused by the collagen nanofibers and β-TCP particles in the scaffold.

Yeo et al.

cells on the composite struts and pores was observed in the hierarchical composite scaffold that consisted of PCL/β-TCP and collagen nanofibers. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffolds was 2.2 times higher than that on the PCL/β-TCP scaffold. In addition, the cell proliferation rate was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we can conclude that the collagen nanofibers and β-TCP particles in the scaffold provided synergistic effects that improved cell activity.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)

4. Conclusions

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In this study, melt-plotted PCL/β-TCP composite scaffolds combined with collagen nanofibers were developed, and various physical properties including water-absorption ability, WCA, and Young’s modulus and cellular activities (e.g., the initial attachment and proliferation of MG63 on these scaffolds) were evaluated. To examine the effects of β-TCP in PCL struts and electrospun collagen fibers, various composite scaffolds with three different compositions of β-TCP (0, 20, 40 wt %) in PCL layered with and without electrospun collagen fibers were fabricated. SEM micrographs of the fabricated scaffold indicated that the β-TCP particles were uniformly embedded in the PCL struts. Additionally, electrospun collagen nanofibers (diameter ) 160 nm) were well layered between the composite struts. Inclusion of β-TCP and collagen nanofibers in the hierarchical scaffold led to dramatic waterabsorption ability (100% increase) and induced hydrophilic properties (20% decrease in WCA). Moreover, the collagen nanofibers in the hierarchical scaffolds did not provide a lowering effect on mechanical properties, as verified by Young’s modulus in the dry state compared to the pure PCL/β-TCP composite. The collagen nanofibers were well sustained in the hierarchical scaffold during the cell-culturing process. SEM images of the cell-scaffold after 7 days of cell culture showed that the most uniform distribution of

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