Annals of Biomedical Engineering ( 2013) DOI: 10.1007/s10439-013-0926-z

Seeding Cells on Calcium Phosphate Scaffolds Using Hydrogel Enhanced Osteoblast Proliferation and Differentiation MIN-HO HONG,2,3 SUNG-MIN KIM,3 JI-YEON OM,3 NAMYONG KWON,4 and YONG-KEUN LEE1 1

YesBioGold Co., Ltd., 227, Moraenae-ro, Seodaemun-gu, Seoul 120-806, Korea; 2Department of Orthopaedic Surgery, Center for Orthopaedic Research, Columbia University Medical Center, 650 West 168th Street, New York, NY 10032, USA; 3 Department of Applied Life Science, Yonsei University College of Dentistry, Seoul 120-752, Korea; and 4SKKU Advanced Institute of Nanotechnology, Sungkyunkwan University, Suwon 440-746, Korea (Received 9 July 2013; accepted 7 October 2013) Associate Editor Smadar Cohen oversaw the review of this article.

Abstract—Internal pores in calcium phosphate (CaP) scaffolds pose an obstacle in cell seeding efficiency. Previous studies have shown inverse relationships between cell attachment and internal pore size, which mainly resulted from cells flowing to the bottom of culture plates. In order to overcome this structure-based setback, we have designed a method for cell seeding that involves hydrogel. CaP scaffolds fabricated with hydroxyapatite, biphasic calcium phosphate, and btricalcium phosphate, had respective porosities of 77.0, 77.9, and 82.5% and pore diameters of 671.1, 694.7, and 842.8 lm. We seeded the cells on the scaffolds using two methods: the first using osteogenic medium and the second using hydrogel to entrap cells. As expected, cell seeding efficiency of the groups with hydrogel ranged from 92.5 to 96.3%, whereas efficiency of the control groups ranged only from 64.2 to 71.8%. Cell proliferation followed a similar trend, which may have further influenced early stages of cell differentiation. We suggest that our method of cell seeding with hydrogel can impact the field of tissue engineering even further with modifications of the materials or the addition of biological factors. Keywords—Bone tissue engineering, Calcium phosphate scaffold, Hydrogel, Cell seeding efficiency, Three-dimensional culture.

INTRODUCTION There are two important factors in the scaffold regarding its efficiency.14 The scaffold must be biocompatible and readily adopted by the biological system, and it has to provide environment where cells can replace damaged tissues effectively without adverse

Address correspondence to Yong-Keun Lee, YesBioGold Co., Ltd., 227, Moraenae-ro, Seodaemun-gu, Seoul 120-806, Korea. Electronic mail: [email protected]

effects. In the past few decades, these two key factors have been researched and developed separately, and it remains unclear what the ideal combination is in maximizing the regeneration of damaged tissue. There are further requirements in the field of hard tissue engineering. For example, interconnected pores and other internal structures conducive to blood penetration and access to nutrients are mandatory for thorough three-dimensional regeneration.32 In the case of a load-bearing, (except in oral and maxillofacial), sufficient mechanical strength is an additional prerequisite for maintaining structural integrity.16 In hard tissue regeneration, calcium phosphate (CaP) materials have been widely used due to their biocompatibility and osteoconductivity. In addition to these characteristics, they are major components of human bone. There are three major CaP materials. Hydroxyapatite (HA, Ca10(PO4)6(OH)2), which makes up most of the hard tissue phase, has excellent biological affinity with host tissue.5 Many in vitro and in vivo studies have provided evidence for its excellent biocompatibility and enhanced healing of bone defects.4,30 However, the non-biodegradable characteristics of HA can cause intrinsic problems, including infections and slow interaction with host cells due to non-bioactivity.9 Beta-tricalcium phosphate (b-TCP, Ca3(PO4)2) is another excellent biodegradable material that can facilitate osteogenesis.7 Although b-TCP is widely used in the clinical field, its high degradation rate can cause problems. Therefore, some recent research has attempted to regulate the degradation rate by changing the calcium to phosphate ratio.24,31 As a result, Biphasic calcium phosphate (BCP), which is more degradable than HA and more stable than bTCP, is also widely used in the clinical field.3

 2013 Biomedical Engineering Society

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Although the biomaterials are biocompatible and bioactive, there is a critical problem regarding cell culture in scaffolds synthesized from these materials.10 Over the past few decades, several strategies on cellbiomaterial interactions have been developed to improve their affinities on porous and three-dimensional (3D) structures.15 Cell seeding efficiency on porous 3D-structured scaffolds is an important factor, because it is difficult for cells to adhere and grow fully on the surface of scaffolds under static and normal cell culture conditions. Therefore, much research has been performed on increasing the cell seeding efficiency through surface modifications and through a variety of cell culture methods (Table 1). In these studies, the cell seeding efficiencies ranged from 25 to 87%. However, highly efficient scaffolds seem to have low porosity or small pore diameters, and their high efficiencies require complex and costly instruments. In this study, hydrogel, which is known to easily encapsulate cells and bioactive molecules, was used.21 The advantages of the hydrogel are as follows: (1) they are biomaterials, which refer to materials of biological origin, (2) they have highly porous structures, including individual nanofibers, (3) it is possible to modify the peptide sequences, and (4) they can integrate into the body without harm. Since the hydrogel has lower mechanical properties such as stress and load bearing compared to the scaffolds, they are not usually applied for bone tissue. Here, we used hydrogel in the cell seeding medium to increase cell seeding efficiency. The hydrogel encapsulated cells were seeded to different types of CaP scaffolds to study the interactions between the cells and the scaffolds. We propose that cell seeding efficiency was increased because hydrogel provided the cell stability. We showed that mixing of hydrogel in the cell seeding medium significantly increased the cell seeding

efficiency on the CaP scaffolds. Furthermore, cell proliferation and differentiation in the early stages without any modification of the materials or the addition of biological factors were also influenced. In this study, we hypothesize that (1) the cell seeding efficiency will increase in all CaP scaffolds when the cells are seeded with hydrogel as compared to seeding using only culture medium, and (2) the initial cell number on the scaffolds will have an indirect influence on cellular behaviors such as proliferation and differentiation. The design of this study is schematically depicted in Fig. 1.

MATERIALS AND METHODS Fabrication and Characterization of Porous CaP Scaffolds CaP scaffolds were prepared from nano-sized HA, BCP, and b-TCP powders (OssGen, Gyeongsan, Korea) using a polymeric template-coating technique as previously reported.19 Briefly, polyurethane sponges were coated with nano-sized CaP powder in a distilled water-based slurry containing 3% polyvinyl alcohol (PVA; Sigma-Aldrich, St. Louis, MO) and 1% methyl cellulose (Sigma-Aldrich, St. Louis, MO), 5% ammonium polyacrylate (OssGen, Gyeongsan, Korea), and 5% N,N-dimethylformamide (Sigma-Aldrich, St. Louis, MO) to improve the sintering driving force and to stabilize the scaffold structure during the sintering process. Slurry coated sponges were dried overnight at 50 C in a drying oven and sintered at 1250 C for 2 h in a furnace (Model BF51314C, Lindberg/Blue, Asheville, NC). The CaP scaffolds were then coated with the slurry again and reinterred under the same conditions to increase their strength. The CaP scaffolds

TABLE 1. Pore properties and cell seeding efficiencies of 3D matrices in previous studies. Porosity (%)

Pore diameter (lm)

Chemical composition

60

100–700

48.8–52.5 – 90 75

780–900 300–600 100–150 400–600

89 95.7 82–94 –

20–50 – 100–200 6.3–13.5

61–63

399–425

Starch with PCL Aliphatic PU PCL-TCP Alginate PEGT/PBT (polyactive) PET PLA/glass PLGA/HA Collagen with PCL/b-TCP PLA

Method

Cell seeding efficiency (%)

References

Gradation of pore size

70

Sobral et al.35

3D bioplotting 3D perfusion culture Surface hydrophilization Perfusion bioreactor system

25–35 69–86 87 69–81

Pfister et al.29 Papadimitropoulos et al.26 Dvir-Ginzberg et al.8 Wendt et al.37

HA exposure on polymeric surface Nanofibers

68 37.6–66.2 62.0–66.5 78

Zhao and Ma38 Koch et al.20 Kim et al.18 Kim and Kim17

Controlled perfusion condition

47.5

Olivares and Lacroix25

Seeding Cells on Calcium Phosphate Scaffolds

FIGURE 1. Representative scheme of this study.

were ground, and their volumes were standardized to 5 9 5 9 5 mm3. The CaP scaffolds were crushed and ground into powder prior to the X-ray diffraction (XRD; Ultima IV, Rigaku, Tokyo, Japan) analysis, which was performed to validate the purity of their compositions. The XRD data were collected using Cu Ka1 radiation and a 2h degree of 20–40. The interconnected pore morphologies of the CaP scaffolds were observed using a stereomicroscope (SZ-6045-TR, Olympus Optical, Tokyo, Japan), and the strut of the scaffolds was observed with scanning electron microscopy (SEM; JSM-6701F, JEOL, Tokyo, Japan). The scaffolds were affixed to the mount with carbon tape and a colloidal carbon adhesive and were pre-coated with a sputtered platinum conductive layer for 150 s. Micro-computed tomography (l-CT; Skyscan 1076, Antwerp, Belgium) analysis was used to determine the apparent porosity and other structural properties of the CaP scaffolds and to observe the architecture at a voltage of 100 kV and a current of 100 lA. All of the CaP scaffolds were scanned under conditions of an xyz resolution of 9.0 lm and an exposure time of 5301 ms. 2D xy slice images were compiled into 3D xyz images, and these 3D architecture images were analyzed to obtain the porosity using a CTAn v.1.12 by 3D visualization software. Atomic force microscopy (AFM) observation of the hydrogel (BDTM PuraMatrixTM, BD Biosciences, USA) was performed to confirm the formation of nanofibers after immersion in the cell culture medium using a SPI4000 Probe Station & SPA-400 SPM Unit (Seiko, Chiba, Japan). The hydrogel was placed on the

single crystalline silicon substrate and air-dried prior to AFM observation. An SI-DF40 tip (Seiko, Chiba, Japan) was used in dynamic force mode cantilevers with a 40 N/m spring constant and a 300 kHz oscillation frequency. Cell Culture and Seeding with a Hydrogel on CaP Scaffolds for Enhanced Cell Seeding Efficiency The MC3T3-E1 mouse calvaria-derived cell line (subclone 4, American Type Culture Collection, Rockville, MD) was cultured, and cell passages between 5–8 were used in the in vitro assays of this study. Cells were cultured at 37 C with 5% CO2 in an incubator and maintained in alpha-modified minimum essential medium (a-MEM; Welgene, Daegu, Korea), supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) and 1% penicillin (Gibco, Grand Island, NY). MC3T3-E1 cells were harvested at subconfluence using a 0.25% trypsin/EDTA solution (Sigma-Aldrich, St. Louis, MO) and were then spun down. The culture medium was removed from the cell pellet, and the cells were resuspended in 10% sucrose and collected again by centrifugation. The CaP scaffolds were incubated in the culture medium for 2 h before cell seeding. The cell seeding efficiency tests were performed in two ways: (1) Control groups; where the 100 lL cell suspension (1 9 106 cells/mL) was seeded into the CaP scaffolds placed in a 24-well plate, and 2) Experimental groups; where the 50 lL cell suspension (2 9 106 cells/mL) and the 50 lL hydrogel were mixed while avoiding bubble formation. The mixture (the final desired cell

HONG et al. TABLE 2. The group codes of this study with their corresponding morphological properties. Control group Group code Raw material Peptide hydrogel Porosity (%) Pore diameter (lm) Strut diameter (lm)

Experimental group

H

B

T

GH

GB

GT

HA 9 77.04 671.12 167.43

BCP 9 77.85 694.74 157.28

b-TCP 9 82.53 842.81 159.14

HA s – – –

BCP s – – –

b-TCP s – – –

H Hydroxyapatite, B Biphasic calcium phosphate, T beta-Tricalcium phosphate, G Peptide hydroGel).

concentration was 1 9 106 cells/mL) was then added to the CaP scaffolds in a 24-well plate (Table 2). Next, 1.5 mL of the culture medium was subsequently added to each well. The scaffolds were placed under standard cell culture conditions for 12 h and were then placed in a fresh 24-well plate to perform the following in vitro assay.35 After 12 h, the cell culture medium was removed (when the medium was removed, vacuum aspiration was not used to avoid the loss of the hydrogel), and the scaffolds and cells were washed twice gently with a phosphate buffered saline (PBS; Gibco, Grand Island, NY). The scaffolds were placed in the new 24-well plate to perform another analysis, and the cells attached to the wells were removed using a 0.25% trypsin/EDTA solution. To count the cell number, automated cell counter (LunaTM, Logos Biosystems, Annandale, VA) was used according the manufacturer’s protocol. The cell seeding efficiency was calculated based on the following equation, which has been generalized in many previous studies.35 Cell seeding efficiency ð%Þ ðinitial seeded cells to scaffold  cells in well plateÞ  100 ¼ initial seeded cells to scaffold

Cell Proliferation Assay The cell proliferation assay was carried out using the Quanti-iTTM PicoGreen dsDNA Reagent and Kits (Invitrogen, Eugene, OR) on days 1, 3, and 7. Briefly, on each day of the experiment, the scaffolds were rinsed 3 times with PBS and placed in a 1.7 mL micro-tube containing TE buffer (10 mM Tris–HCl, 1 mM EDTA). The scaffolds were stored in a freezer at 280 C. After thawing, they were vortexed for 10 s to extract the cellular DNA from the matrix. The solution was centrifuged at 12,000 rpm for 10 min at 4 C to collect the supernatants for assay. The supernatants were then placed into individual wells of a black 96well plate, and PicoGreen dsDNA reagents were subsequently placed into the well in the same ratio. The plate was allowed to incubate for 5 min in the dark at

room temperature, and the fluorescence was measured on a microplate reader (POLARstar OPTIMA MicroPlate Reader, BMG Labtech, Offenburg, Germany) using an excitation wavelength of 485 nm and an emission wavelength of 520 nm. A standard curve was plotted using the provided kDNA standard. Acetoxymethyl Ester of Calcein Staining for Cellular Viability Measurements After 1 day, the initial cell viability of MC3T3-E1 in the pure hydrogel, control group, and experimental group were observed under a fluorescent microscope using acetoxymethyl ester of calcein (calcein AM; Live/ Dead Viability/Cytotoxicity Kit, Invitrogen, Carlsbad, CA) staining. Briefly, the cell culture medium was removed gently from each cell insert using a micropipette to protect the relatively soft fibrous matrix, and the adherent cells on the samples were washed with Dulbecco’s phosphate buffered saline (DPBS; Gibco, Grand Island, NY). The aqueous solution of calcein AM was added to all samples and incubated for 30 min at 37 C. After incubation, the solution was removed, and DPBS was added to prevent the samples from drying out. The labeled cells were visualized under a confocal laser scanning microscope (LSM 700, Carl Zeiss, Ostalbkreis, Germany). Cytoskeleton Staining and Observation After 2 weeks of seeding cells on the scaffolds, the actin formation on the scaffolds was observed by staining the actin filaments and the nuclei of MC3T3E1 using phalloidin and 4¢,6-diamidino-2-phenylindole (DAPI), respectively. For actin staining, the scaffolds were gently rinsed three times with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Permeabilization was performed with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 5 min, and blocking was carried out in a 1% bovine serum albumin for 30 min. F-actin filaments were stained with fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma-Aldrich, St. Louis, MO) in PBS (50 lg/mL)

Seeding Cells on Calcium Phosphate Scaffolds

and incubated for 1 h. The scaffolds were then washed twice with PBS, and the nuclei were co-stained using Prolong gold anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA) to preserve the fluorescence for 5 min. The scaffold fluorescence imaging was performed using the aforementioned microscope. ALP Activity Enzyme Assay The ability of MC3T3-E1 differentiation was evaluated by the alkaline phosphatase (ALP) activity, which is considered as an early marker for osteoblast differentiation.14 After 1, 2, and 3 weeks of cell seeding, the ALP activity of the MC3T3-E1 cells in the scaffolds was measured with a Sensolyte p-nitrophenyl phosphate (pNPP) Alkaline Phosphatase Assay Kit (AnaSpec, Fremont, CA), where the ALP activity assay was performed according to the manufacturer’s protocol. Briefly, the cell seeded scaffolds were gently washed twice with PBS, and the scaffolds were placed into a 1.7 mL micro tube. One milliliter of 0.2% Triton X-100 was added to the micro tube for cell lysis, and each tube was vigorously vortexed for 30 s. The cell suspension was incubated at 4 C for 10 min on the rotator and centrifuged at 25009g for 10 min at 4 C. Aliquots with volumes of 50 lL from cell lysates were placed into a 96-well microplate and mixed with the same amount of the pNPP working solution. The reaction of the mixtures was carried out at room temperature for 2 h, and the absorbance was measured at 405 nm using a spectrophotometric plate reader (Epoch, BioTek, Winooski, VT). The ALP activity was normalized using the total protein amounts by the Bradford method (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA).6

with a thermocycler (2720 thermal cycler, Applied Biosystems, Foster City, CA). Real time PCR analysis was performed using Taqman Gene Expression Assay (Applied Biosystems, Foster City, CA) and as the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Mm99999915_g1) was applied as with the other experiments. In this study, type I collagen (COL-I, Mm00483888_m1), osteocalcin (OCN, Mm03413826_mH), and runt-related transcription factor 2 (RUNX2, Mm00501584_m1) from Taqman PCR primers were selected for measurement of osteoblast differentiation. cDNA sample (2 lL) of each scaffold was mixed with each primer (1 lL) and Taqman Universal Master Mix II with UNG (10 lL). The final mixtures in the optical 96-well reaction plate (MicroAmpTM, Applied Biosystems, Foster City, CA) were quantified with the following condition: 50 C for 2 min, 95 C for 10 min, 40 cycles at 95 C for 15 s, and 60 C for 1 min using the 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA). Each gene expression level was calculated according to relative quantification values and they were normalized to the level of expression of the cells seeded on the culture dish.33 Relative quantification of target genes was calculated using the 22ddCT (CT; cycle threshold) method.

Statistical Analysis All data were expressed as the mean ± the standard deviation, and they were compared using t test and one-way ANOVA, followed by Tukey’s comparison post hoc analysis using PASW Statistics 18 (SPSS, Chicago, IL). Significant differences between each group were determined at p < 0.05 (n = 3).

Extraction of RNA and Real-Time PCR After 3 weeks of cell seeding, total RNA of the cells on the scaffolds were extracted using Trizol (SigmaAldrich, St. Louis, MO) according to the routine protocol and reverse-transcribed into cDNA using Omniscript RT kit (Qiagen, Valencia, CA). Briefly, cells on the scaffolds were immersed in 200 lL of Trizol and the scaffolds were crushed. After 5 min, chloroform (Amresco, Solon, OH) was added and the mixture was centrifuged for phase separation of RNA and DNA. The separated RNA was precipitated with isopropyl alcohol (Amresco, Solon, OH) and then dissolved in RNase-free water after washing with 75% ethanol. The reverse transcription mixture was prepared with 3 lL of 109 buffer RT, 3 lL of dNTP mix, 1.5 lL of Oligo-dT primers, and OmniscriptTM RT. The final volume of mixture was adjusted to 30 lL and the mixture was thermal cycled at 37 C for 90 min

RESULTS Chemical and Morphological Characteristics of the Scaffolds Three different scaffolds were fabricated by the same method but with different raw materials. XRD analysis, which is a basic and essential analytical technique, was performed on each scaffold to identify intrinsic crystalline structures. The results showed typical patterns for each scaffold (Fig. 2a). Optical images were obtained using a stereomicroscope as shown in Fig. 2b. The images show that the pore structures of the scaffolds contained significant interconnectivity, and the 3D conversion l-CT images in Fig. 2c show the interconnected pores in tandem. Micro-CT analysis corroborated the structural

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properties of the scaffolds, including porosity, pore size, and strut size (Table 2). The struts of each scaffold were observed using SEM (Fig. 2d), and their images showed highly fused particles post sintering. The hydrogel was composed of a peptide that selfassembles into a 3D structure under physiological conditions. The nanofiber-structured hydrogels were observed using AFM, as represented in Fig. 3, showing dozens of nano-sized fibers and hundreds of nano-sized pores. These characteristics were verified before it was used to increase the cell seeding efficiency on the scaffolds.22 Cell Attachment on the 3D Composite Matrix MC3T3-E1 cells were seeded on the three different scaffolds using two different cell seeding methods, and the cell seeding efficiency was calculated by the aforementioned equation. The results of the cell seeding efficiency are presented as a column in Fig. 4a. The efficiency of experimental group was between 92.5– 96.3%, while control group was between 64.2–71.8%. There were significant differences between the experimental group and the control group in each scaffold. After 1 day of cell seeding, live cells on the scaffolds and in the hydrogel were stained with calcein AM. While the control group showed that the cells are

attached on the strut, cells were sustained with hydrogel in experimental group. Fluorescence Imaging for the Observation of Cell Spreading and Migration Cell proliferation and cell spreading on the scaffold, along with migration in the hydrogel, were observed with actin filaments and nuclei staining on the scaffolds 2 weeks after seeding (Fig. 5). The cells on the scaffolds with well-developed actin filaments can be observed in the magnified images in each figure. As shown in the upper images, the strut of the scaffolds was covered completely by seeded cells under the general method. The lower images show the cells in the hydrogel and the strut of the scaffolds covered by cells. It may imply that the cells in hydrogel migrated onto the strut after 2 weeks. Cell Proliferation and Differentiation Cell proliferation on scaffolds with different seeding methods was analyzed with the PicoGreen dsDNA assay. The DNA quantification of each group was calibrated by a kDNA curve. As shown in Fig. 6a, the cell proliferation levels of all groups increased with culture time demonstrating that the

FIGURE 2. (a) XRD patterns of HA, BCP, and b-TCP scaffolds showing their characteristic peaks in the range of 20–40°. (b) Digital photographs of each scaffold show that their pores are interconnecting, and (c) the images of l-CT verify their interconnectivity and also help determine their porosity. (d) SEM images showing the skeletons of each scaffold, and that there was no visible difference between samples (The inserts in the upper right corner of the images are magnified 35000).

Seeding Cells on Calcium Phosphate Scaffolds

FIGURE 3. (a) Typical AFM images showing the nanofibers and the pores. (b) Tilted image to observe the height of the fibers.

FIGURE 4. (a) The cell seeding efficiency of each scaffold is expressed using columns. The statistical analysis was performed by t test to compare between the same CaP used scaffolds. There were significant differences in all statistical analyses. (b) Confocal microscope images of each scaffold showing that the number of live cells in the groups cell seeded with the hydrogel was higher than those without the hydrogel.

cells in the hydrogel were not affected critically by the type of CaP scaffold. However, the DNA concentrations in the experimental groups were higher than in the control groups. Additionally, there were significant differences between the experimental group and the control group (p < 0.05) showing that cell seeding with the hydrogel was more advantageous for the initial cell adhesion and proliferation. While cells in GH and GT groups showed early proliferation, cells in GB group started to proliferate after day 3. Quantification of the ALP activity was performed after 7, 14, and 21 days using a colorimetric pNPP based reaction for initial osteoblast differentiation. As shown in Fig. 6b, the ALP activity of all the groups increased substantially with culture time as with the results of the cell proliferation. There were also significant differences between the control group and the experimental group in days 7, 14, and 21 when the result was analyzed by one-way ANOVA (p < 0.05). The ALP activity of the cells on the scaffolds without the hydrogel showed a distinct increase after 2 weeks,

while the ALP activity of experimental groups increased constantly from the first day of the experiment. However, when the results were analyzed between the same CaP used scaffolds by t test, there was no significant difference except the b-TCP used groups after 21 days. Notably, the GH group had the highest ALP activity compared with the other groups. After 3 weeks from seeding cells on the scaffolds, each mRNA expression level of RUNX2, COL-I, and OCN was calculated by real-time PCR (Fig. 7). RUNX2 was expressed at experimental day, however it seemed that the expression level was neither affected by time nor cell seeding methods. As COL-I expression levels of each group, there was no obvious difference between each group unlike the results of previous in vitro assays, however the expression levels in the group GH were the highest at all experimental days, and showed significant difference with other groups. As the expression levels of OCN, there was no significant difference between each group. Consequently, the each level of groups was not cell seeding methoddependent critically.

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FIGURE 5. Cytoskeleton (green) and nuclei (blue) stained MC3T3-E1 cells after 14 days of seeding. The upper images (groups H, B, and T) show that the skeleton of the scaffolds was thoroughly covered by cells, as expected. The lower images (groups GH, GB, and GT) show that the cells migrated from the gel to the surface of the skeleton. The inserts are magnified images showing the actin fibers more accurately. (Some dark areas of the skeleton are out of focus and may be covered by cells.).

FIGURE 6. (a) The cell proliferation of MC3T3-E1 cultured on the CaP scaffolds according to the different methods for 7 days as assessed by PicogreenÒ dsDNA assays. (b) The ALP activity of MC3T3-E1 cultured on the 3D scaffold systems at 7, 14, and 21 days. Significant differences between the groups were expressed by special letters (*; GH and GB, „ ; GB, #; GH, and –; H.).

DISCUSSION Different strategies for bone tissue engineering have been developed in steady improvements in cellular response. Generally, these strategies have modified the materials, surface characteristics, structural properties, and mechanical properties but not limited to modifications in types of cells, culture mediums, growth factors, and culture conditions.28,36 In this study, the three different CaP scaffolds, which have been widely used in the field of bone tissue engineering, were used

as basic materials. The main variable of this study was the seeding method. The BDTM PuraMatrixTM hydrogel used in this study has already been used in numerous papers, and there are many different protocols for cases specific to cell culturing conditions using this hydrogel (BD Biosciences PuraMatrix product information sheet, www.puramatrix.com).12,23 Many scaffolds for tissue engineering have been developed using various raw materials that exhibit a variety of pore properties. The pore properties are known to be factors that are inversely proportional to

Seeding Cells on Calcium Phosphate Scaffolds

FIGURE 7. The results of real-time PCR for RUNX2, COL-I, and OC measured in MC3T3-E1 after 21 days cultured on the scaffolds. Significant differences between the groups were expressed by special letters (*; GH, „ ; T and B, #; B and GB).

the mechanical properties. However, it is also known that an increased pore size and porosity are beneficial for the migration of cells and nutrients, as well as for vascularization.21 The increased pore size and high porosity are advantageous to cellular behaviors, but they can also cause problems in the initial stages of cell cultures on the scaffolds. When cells in the medium are seeded on the scaffolds, some cells adhere to the skeleton but others flow down to the bottom of the well plate. This is due to the morphology of the scaffolds and the water-like culture medium, which is more similar to the viscosity of water than that of blood plasma. The viscosity of the hydrogel used in this study can be reduced by vortexing. The hydrogel with decreased viscosity readily returned to the gel state under physiological conditions, which then could be applied to the CaP scaffolds to increase the cell seeding efficiency. Three different CaP materials were used to fabricate the scaffolds, but their pore properties remained

similar, because the pore properties could be critical parameters that might affect the results of this study. The three types of scaffolds had their own crystalline phases, but their shapes and pore properties of each scaffold were similar (Fig. 2 and Table 2). Based on these results, in vitro assays were performed under the hypothesis that the raw materials of the scaffolds were different but that their morphological properties were similar to each other. Cell seeding efficiency was calculated using an automated cell counter, and Fig. 4a shows the cell seeding efficiency for each group. These results indicate that the use of the hydrogel for cell seeding significantly improved the cell seeding efficiency. Considering that the porosities of the scaffolds were similar, it can be concluded that the number of cells seeded on the scaffolds with the hydrogel was higher than the number of cells seeded on the scaffolds under normal cell culture medium conditions. Because of the large voids (interconnected pores that occupy approximately 80%

HONG et al.

of total scaffold volume) cells in water-like culture media may flow down with gravity. However, hydrogel is entrapped in the pores of the CaP scaffolds due to its high viscosity and self-assembling properties under physiological conditions. The upper images in Fig. 4b show that the hydrogel containing live cells was entrapped in the interconnected pores of the scaffolds, while the groups without the hydrogel showed cells only on the surface of the strut, as observed in previous studies (lower images in Fig. 4b). Cell proliferation was quantified by DNA concentration. These results seemed consistent with the results of cell seeding efficiency. Compared to the control group, the number of initial adherent cells was significantly higher, which then further increase the proliferation rate. It may seem that the different proliferation rates between the control groups and experimental groups were due to the initial number of attached cells. The relationship between the initial cell number and the cell proliferation has been previously reported.27,28 ALP activity indicates the level of cell differentiation at an early stage. In our study, the ALP activity was measured on days 7, 14, and 21. The cell seeding results were similar to those presented in the in vitro assay results, which showed their tendency to increase over time. There were also significant differences between the control groups and experimental groups. However, the level of cell differentiation in the control group increased drastically on day 21. The aforementioned conjecture was confirmed through the results of real time PCR. According to the expression levels of RUNX2, which has been identified as a bone-specific transcription factor, there was no significant difference between each group. Based on these results, the results of ALP activity on the day 21 could be understood, besides the expression levels of COL-I, which is also known as an early bone marker gene, showed the only slight differences between each group, but it seemed that there was no significant connection between the cell number and the cell differentiation level. The expression levels of OCN, which is known as a late differentiation marker gene, there was also no significant difference between each group. The results of OCN also confirmed that the cell number could not significantly affect the cell differentiation in late stage. The results of in vitro tests showed that high cell seeding efficiency by hydrogel exerted a good influence on cell proliferation and differentiation at an early stage. However, there were significant differences among control groups and experimental groups. The results of HA used groups were higher than other groups, because the biodegradability can effect on the

static in vitro environment. If in vitro tests are progressed using bioreactor, different results can be expected.13,34 In this study, the high number of cells on the scaffolds with hydrogel showed that the initially attached cells could influence the cellular behavior up to the early stages of differentiation. Although the cell seeding method with the hydrogel was not established until the mid to late stages of cell differentiation, we can design experimental protocols for bone tissue engineering by the addition of biological factors in the hydrogel and modification of the sequence of the peptide. There have been many similar studies using the hydrogel.14,22 The method of cell seeding on the CaP scaffolds with the hydrogel also has an advantage of the cells proliferating and differentiating under real 3D conditions. The 3D cultures on CaP scaffolds can be thought of as having 3D conditions, but the cells on the strut experience 2D conditions. Previous studies have used 3D culture condition to mimic in vivo as the culture conditions can influence cell morphology as well as specific gene expression.1,2,11 As a result, we can conclude that this method of cell seeding on CaP scaffolds with hydrogel has infinite possibilities in the field of bone tissue engineering. We have designed a cell seeding method on CaP scaffolds using hydrogel that can significantly increase cell seeding efficiency, while also influencing cell proliferation and differentiation at an early stage. The CaP scaffolds used in this study had a porosity high enough and a pore size large enough for blood to infiltrate the scaffold and the cells to migrate. In this study, we controlled cellular behavior without modification of the substrate (scaffolds) and additional biological factors. From this study, we were able to conclude that cell seeding on scaffolds with hydrogel has the potential to become a standard method widely used with further potential modification of the materials and the addition of biological factors (e.g., vascular endothelial growth factor, bone morphogenetic protein, insulin growth factor, fibroblastic growth factor, etc.).

ACKNOWLEDGMENTS We thank Heon Goo Lee (Columbia University, NY), Jaeryong Ko (Vassar College, NY), Phillip Lim (Johns Hopkins University, MD), Jae-Sung Kwon, M.D. (Yonsei University, Korea), and Kang-Sik Lee, Ph.D. (ASAN Medical Center, Korea) for their helpful comments.

Seeding Cells on Calcium Phosphate Scaffolds

REFERENCES 1

Abbott, A. Cell culture: biology’s new dimension. Nature 424(6951):870–872, 2003. 2 Cukierman, E., R. Pankov, D. R. Stevens, and K. M. Yamada. Taking cell-matrix adhesions to the third dimension. Science 294(5547):1708–1712, 2001. 3 Daculsi, G. Biphasic calcium phosphate concept applied to artificial bone, implant coating and injectable bone substitute. Biomaterials 19(16):1473–1478, 1998. 4 Damien, E., K. Hing, S. Saeed, and P. A. Revell. A preliminary study on the enhancement of the osteointegration of a novel synthetic hydroxyapatite scaffold in vivo. J. Biomed. Mater. Res. A 66A(2):241–246, 2003. 5 Degroot, K. Bioceramics consisting of calcium-phosphate salts. Biomaterials 1(1):47–50, 1980. 6 Delgado-Calle, J., C. Sanudo, L. Sanchez-Verde, R. J. Garcia-Renedo, J. Arozamena, and J. A. Riancho. Epigenetic regulation of alkaline phosphatase in human cells of the osteoblastic lineage. Bone 49(4):830–838, 2011. 7 Dorozhkin, S. V., and M. Epple. Biological and medical significance of calcium phosphates. Angew. Chem. Int. Ed. 41(17):3130–3146, 2002. 8 Dvir-Ginzberg, M., I. Gamlieli-Bonshtein, R. Agbaria, and S. Cohen. Liver tissue engineering within alginate scaffolds: effects of cell-seeding density on hepatocyte viability, morphology, and function. Tissue Eng. 9(4):757–766, 2003. 9 Hattori, H., K. Masuoka, M. Sato, M. Ishihara, T. Asazuma, B. Takase, M. Kikuchi, K. Nemoto, and M. Ishihara. Bone formation using human adipose tissue-derived stromal cells and a biodegradable scaffold. J. Biomed. Mater. Res. B 76B(1):230–239, 2006. 10 Hench, L. L., and J. M. Polak. Third-generation biomedical materials. Science 295(5557):1014–1017, 2002. 11 Hoffman, R. M. To do tissue-culture in 2 or 3 dimensions—that is the question. Stem Cells 11(2):105–111, 1993. 12 Holmes, T. C., S. de Lacalle, X. Su, G. S. Liu, A. Rich, and S. G. Zhang. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proc. Natl Acad. Sci. U.S.A. 97(12):6728–6733, 2000. 13 Holtorf, H. L., T. L. Sheffield, C. G. Ambrose, J. A. Jansen, and A. G. Mikos. Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics. Ann. Biomed. Eng. 33(9):1238–1248, 2005. 14 Horii, A., X. M. Wang, F. Gelain, and S. G. Zhang. Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration. PLoS ONE 2(2):e190, 2007. 15 Hubbell, J. A. Biomater. Tissue Eng. 13(6):565–576, 1995. 16 Hutmacher, D. W. Scaffolds in tissue engineering bone and cartilage. Biomaterials 21(24):2529–2543, 2000. 17 Kim, Y. B., and G. Kim. Rapid-prototyped collagen scaffolds reinforced with PCL/beta-TCP nanofibres to obtain high cell seeding efficiency and enhanced mechanical properties for bone tissue regeneration. J. Mater. Chem. 22(33):16880–16889, 2012. 18 Kim, S. S., M. S. Park, O. Jeon, C. Y. Choi, and B. S. Kim. Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials 27(8):1399– 1409, 2006. 19 Kim, S. M., S. A. Yi, S. H. Choi, K. M. Kim, and Y. K. Lee. Gelatin-layered and multi-sized porous beta-tricalcium phosphate for tissue engineering scaffold. Nanoscale Res. Lett. 7(1):78, 2012.

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Koch, M. A., E. J. Vrij, E. Engel, J. A. Planell, and D. Lacroix. Perfusion cell seeding on large porous PLA/calcium phosphate composite scaffolds in a perfusion bioreactor system under varying perfusion parameters. J. Biomed. Mater. Res. A 95A(4):1011–1018, 2010. 21 Laurencin, C. T. N. L. S. Nanotechnology and Tissue Engineering: The Scaffold. Boca Raton: CRC Press, 2008. 22 McGrath, A. M., L. N. Novikova, L. N. Novikov, and M. Wiberg. BDTM PuraMatrixTM peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration. Brain Res. Bull. 83(5):207–213, 2010. 23 Narmoneva, D. A., O. Oni, A. L. Sieminski, S. G. Zhang, J. P. Gertler, R. D. Kamm, and R. T. Lee. Self-assembling short oligopeptides and the promotion of angiogenesis. Biomaterials 26(23):4837–4846, 2005. 24 Nery, E. B., R. Z. Legeros, K. L. Lynch, and K. Lee. Tissue-response to biphasic calcium-phosphate ceramic with different ratios of Ha/Beta-Tcp in periodontal osseous defects. J. Periodontol. 63(9):729–735, 1992. 25 Olivares, A. L., and D. Lacroix. Simulation of cell seeding within a three-dimensional porous scaffold: a fluid-particle analysis. Tissue Eng. C 18(8):624–631, 2012. 26 Papadimitropoulos, A., S. A. Riboldi, B. Tonnarelli, E. Piccinini, M. A. Woodruff, D. W. Hutmacher, and I. Martin. A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion. J. Tissue Eng. Regen. Med. 7(3):183–191, 2013. 27 Park, J., S. Bauer, K. A. Schlegel, F. W. Neukam, K. von der Mark, and P. Schmuki. TiO2 nanotube surfaces: 15 nm—an optimal length scale of surface topography for cell adhesion and differentiation. Small 5(6):666–671, 2009. 28 Park, J., S. Bauer, P. Schmuki, and K. von der Mark. Narrow window in nanoscale dependent activation of endothelial cell growth and differentiation on TiO2 nanotube surfaces. Nano Lett. 9(9):3157–3164, 2009. 29 Pfister, A., R. Landers, A. Laib, U. Hubner, R. Schmelzeisen, and R. Mulhaupt. Biofunctional rapid prototyping for tissue-engineering applications: 3D bioplotting versus 3D printing. J. Polym. Sci. A 42(3):624–638, 2004. 30 Rose, F. R., L. A. Cyster, D. M. Grant, C. A. Scotchford, S. M. Howdle, and K. M. Shakesheff. In vitro assessment of cell penetration into porous hydroxyapatite scaffolds with a central aligned channel. Biomaterials 25(24):5507– 5514, 2004. 31 Ryu, H. S., H. J. Youn, K. S. Hong, B. S. Chang, C. K. Lee, and S. S. Chung. An improvement in sintering property of beta-tricalcium phosphate by addition of calcium pyrophosphate. Biomaterials 23(3):909–914, 2002. 32 Sanchez-Salcedo, S., A. Nieto, and M. Vallet-Regi. Hydroxyapatite/beta-tricalcium phosphate/agarose macroporous scaffolds for bone tissue engineering. Chem. Eng. J. 137(1):62–71, 2008. 33 Schmittgen, T. D., B. A. Zakrajsek, A. G. Mills, V. Gorn, M. J. Singer, and M. W. Reed. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal. Biochem. 285(2):194–204, 2000. 34 Schumacher, M., F. Uhl, R. Detsch, U. Deisinger, and G. Ziegler. Static and dynamic cultivation of bone marrow stromal cells on biphasic calcium phosphate scaffolds derived from an indirect rapid prototyping technique. J. Mater. Sci. Mater. Med. 21(11):3039–3048, 2010. 35 Sobral, J. M., S. G. Caridade, R. A. Sousa, J. F. Mano, and R. L. Reis. Three-dimensional plotted scaffolds with controlled pore size gradients: effect of scaffold geometry on

HONG et al. mechanical performance and cell seeding efficiency. Acta Biomater. 7(3):1009–1018, 2011. 36 Wang, H. Y., D. T. K. Kwok, M. Xu, H. G. Shi, Z. W. Wu, W. Zhang, and P. K. Chu. Tailoring of mesenchymal stem cells behavior on plasma-modified polytetrafluoroethylene. Adv. Mater. 24(25):3315–3324, 2012. 37 Wendt, D., A. Marsano, M. Jakob, M. Heberer, and I. Martin. Oscillating perfusion of cell suspensions through

three-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnol. Bioeng. 84(2):205–214, 2003. 38 Zhao, F., and T. Ma. Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development. Biotechnol. Bioeng. 91(4):482–493, 2005.