Acta Biomaterialia xxx (2016) xxx–xxx
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Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering q Sang Jin Lee a,b, Donghyun Lee b, Taek Rim Yoon c, Hyung Keun Kim c, Ha Hyeon Jo a, Ji Sun Park a, Jun Hee Lee a, Wan Doo Kim a, Il Keun Kwon b,⇑,1, Su A Park a,⇑,1 a
Department of Nature-Inspired Nanoconvergence Systems, Korea Institute of Machinery and Materials, 156 Gajeongbuk-ro, Yuseong-gu, Daejeon 304-343, Republic of Korea Department of Dental Materials, School of Dentistry, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701, Republic of Korea c Center for Joint Diseases, Chonnam National University Hwasun Hospital, 322 Seoyang-ro, Hwasun-eup, Hwasun-gun, Jeonnam 519-809, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 11 November 2015 Received in revised form 21 January 2016 Accepted 5 February 2016 Available online xxxx Keywords: Polycaprolactone 3D-printing Recombinant human bone morphogenic protein-2 Bone tissue engineering
a b s t r a c t For tissue engineering, a bio-porous scaffold which is applied to bone-tissue regeneration should provide the hydrophilicity for cell attachment as well as provide for the capability to bind a bioactive molecule such as a growth factor in order to improve cell differentiation. In this work, we prepared a three-dimensional (3D) printed polycaprolactone scaffold (PCLS) grafted with recombinant human bone morphogenic protein-2 (rhBMP2) attached via polydopamine (DOPA) chemistry. The DOPA coated PCL scaffold was characterized by contact angle, water uptake, and X-ray photoelectron spectroscopy (XPS) in order to certify that the surface was successfully coated with DOPA. In order to test the loading and release of rhBMP2, we examined the release rate for 28 days. For the In vitro cell study, pre-osteoblast MC3T3-E1 cells were seeded onto PCL scaffolds (PCLSs), DOPA coated PCL scaffold (PCLSD), and scaffolds with varying concentrations of rhBMP2 grafted onto the PCLSD 100 and PCLSD 500 (100 and 500 ng/ml loaded), respectively. These scaffolds were evaluated by cell proliferation, alkaline phosphatase activity, and real time polymerase chain reaction with immunochemistry in order to verify their osteogenic activity. Through these studies, we demonstrated that our fabricated scaffolds were well coated with DOPA as well as grafted with rhBMP2 at a quantity of 22.7 ± 5 ng when treatment with 100 ng/ml rhBMP2 and 153.3 ± 2.4 ng when treated with 500 ng/ml rhBMP2. This grafting enables rhBMP2 to be released in a sustained pattern. In the in vitro results, the cell proliferation and an osteoconductivity of PCLSD 500 groups was greater than any other group. All of these results suggest that our manufactured 3D printed porous scaffold would be a useful construct for application to the bone tissue engineering field. Statement of Significance Tissue-engineered scaffolds are not only extremely complex and cumbersome, but also use organic solvents which can negatively influence cellular function. Thus, a rapid, solvent-free method is necessary to improve scaffold generation. Recently, 3D printing such as a rapid prototyping technique has several benefits in that manufacturing is a simple process using computer aided design and scaffolds can be generated without using solvents. In this study, we designed a bio-active scaffold using a very simple and direct method to manufacture DOPA coated 3D PCL porous scaffold grafted with rhBMP2 as a means to create bone-tissue regenerative scaffolds. To our knowledge, our approach can allow for the generation of scaffolds which possessed good properties for use as bone-tissue scaffolds. Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
q
Part of the Special Issue on Zwitterionic Materials, organized by Professors Shaoyi Jiang, Kazuhiko Ishihara, and Jian Ji.
⇑ Corresponding authors at: Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 26 Kyungheedaero, Dongdaemun-gu, Seoul 130-701, Republic of Korea (I.K. Kwon), fax: +82 42 868 7933 (S.A. Park). E-mail addresses:
[email protected] (I.K. Kwon),
[email protected] (S.A Park). 1 Two corresponding authors equally contributed to this work. http://dx.doi.org/10.1016/j.actbio.2016.02.006 1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
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S.J. Lee et al. / Acta Biomaterialia xxx (2016) xxx–xxx
1. Introduction To date in the osteogenesis-related field, biomedical researchers have developed three-dimensional (3D) bio-scaffolds for use as implants into a defective area for recovery of lost bone tissue. These scaffolds are biodegradable allowing for resorption in the human body after several months to a year [1]. In order to induce rapid bone tissue regeneration, the bone scaffold should possess porous, lm-sized structure which provides a good environment for cell growth [2–5]. For this reason, several techniques have been developed for creating porous scaffolds including sponge impregnation techniques [6], salt leaching and solvent casting [7], particle-leaching [8], ultra-sonication [9], and freeze-drying [10]. Unfortunately, these processes are not only extremely complex and cumbersome, but also use organic solvents which can negatively influence cellular proliferation and differentiation. Thus, a rapid, solvent-free method is necessary to improve scaffold generation. Recently, 3D printing has received a great deal of attention in the bio-engineering field. This system has several benefits in that manufacturing is a simple process using computer aided design (CAD) and scaffolds can be generated without using solvents [11,12]. 3D printing also allows for manufacturing scaffolds with controlled-pore structures as well as with an overall shape that matches the anatomical defect. This can be accomplished using computer data collected from tomography (CT) and magnetic resonance imaging (MRI) to create a scaffold that matches the patient’s defect site [13]. Because of these advantages, 3D printing systems, such as a rapid prototyping techniques, have been well employed in fields of research related to bone tissue engineering [14,15]. Our previous work described the use of 3D-printed polycaprolactone (PCL) scaffold to promote bone tissue regeneration [16,17]. Typically 3Dprinted scaffolds are generated by synthetic polymers such as polycaprolactone. However, these polymers have poor surface properties and do not have effective chemical functional groups for cell growth and proliferation [18,19]. In order to solve this issue, many biomedical researchers have modified the surfaces of tissue-engineered PCL scaffolds using techniques such as plasma deposition [20], starch-blending [21], or attachment of mussel inspired material [22]. One facile strategy is the use of mussel-inspired chemistry such as polydopamine (DOPA). This has been successfully used in various bio-engineering fields in order to overcome the limitations of synthetic polymers. Dopamine has abundant catechol and amine groups which can coat DOPA layers onto any substrate by just raising the pH without using organic solvents [23]. DOPA can also be coupled with bioactive molecules containing primary amine or thiol groups [24,25]. Herein, we designed a bio-active scaffold using a very simple and direct method to manufacture DOPA-coated 3D PCL porous scaffold grafted with rhBMP2 as a means to create bone-tissue regenerative scaffolds. The rhBMP2 was used in this work because it functions to promote rapid bone healing [26]. The goal of this study is to effectively deliver rhBMP2 in a sustained manner via DOPA chemistry, thereby giving a favorable environment for cellular proliferation and osteogenic differentiation. A schematic illustration of our scaffold generation method is depicted in Fig. 1. The manufactured products were evaluated by chemical–physical characterization followed by in vitro assessment. 2. Materials and methods 2.1. Materials Polycaprolactone (MW 45 kDa), TrizmaÒ hydrochloride, and 3hydroxytyramine hydrochloride (dopamine hydrochloride) were
purchased from Sigma–Aldrich (St. Louis, MO, USA). The Escherichia coli-expressed rhBMP2 was purchased from GENOSS Co., Ltd. (Suwon, Korea). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (PBS), fetal bovine serum (FBS), trypsin–EDTA, and penicillin streptomycin were purchased from Gibco BRL (Invitrogen Co. Ltd, Carlsbad, CA, USA). Osteogenic medium used in this study was comprised of DMEM containing 10% FBS, 1% penicillin–streptomycin, 10 mM b-glycerol phosphate disodium salt hydrate (Sigma–Aldrich, St. Louis, MO), 300 lM ascorbic acid (Sigma) and 0.1 lM dexamethasone (Sigma). Deionized–distilled water (DDW) was produced by an ultrapure water system (Puris-Ro800; Bio Lab Tech., Korea). All other reagents and solvents were of analytical grade and used without further purification.
2.2. Fabrication of the 3D-printed PCL scaffold The PCL scaffold was prepared using a 3D bio-printing system (laboratory made system in Korea Institute of Machinery and Materials). The printing instrumentation consisted of a 3D bioprinting system equipped with a three-axis x–y–z translation stage, dispenser, nozzle, compression/heat controller, and software system. The dispenser was covered with a heating jacket to melt the PCL polymer. The PCL pellets were melted at 80 °C in a heating dispenser, and 3D plotting strands were made using the 3D printing system. When the PCL was melted, a continuous air pressure of 300 kPa was applied to the dispenser which extruded the 3D strand and this was plotted layer-by-layer. The nozzle size was 400 lm and the strand distance was 400 lm in a square pattern. 2.3. Preparation of DOPA coated PCL scaffolds (PCLSDs) and grafting of rhBMP2 3D PCL scaffolds were immersed in dopamine hydrochloride solution (2 mg/mL in 10 mM of Tris buffer, pH 8.5) at room temperature. After 2 h, the DOPA-coated 3D PCL scaffolds were washed with fresh deionized water as three times and dried under nitrogen gas. After that, rhBMP2 was directly grafted by immersing the PCLSD in rhBMP2 solution (100 ng/ml and 500 ng/ml in 10 mM of Tris buffer, pH 8.5) with stirring for 24 h at room temperature. After immobilization of rhBMP2, outcomes were also washed with fresh deionized water as three times. The rhBMP2 grafted PCLSD scaffolds (100 and 500 ng/ml) were abbreviated as PCLSD 100 and PCLSD 500 in this study.
2.4. Release measurement and quantification of the grafting content of rhBMP2 grafted PCL scaffold In order to certify the loading amount and release of grafted rhBMP2 from scaffold, the PCLSD 500 was incubated in 1 ml of PBS at 37 °C for up to 28 days under orbital shaking with 100 rpm. A standard curve was generated using known concentrations of rhBMP2 (1–500 ng/ml). The release was determined at predetermined time intervals of 1, 3, 5, 7, 14, 21, and 28 days. At each time point the entire supernatant was removed from the vial and refreshed with fresh PBS. The collected supernatants were used as specimens for measuring the release rate of rhBMP2. For quantification of the grafting quantity of rhBMP2 on the scaffolds, the supernatants from the loading of PCLSD 100 and PCLSD 500 were collected and the amount of unattached rhBMP2 was quantified. The quantity of rhBMP2 in each test was determined by ELISA using an ELISA kit per manufacturer’s instructions (900-K255, 900-K00, Peprotech Inc., Rocky Hill, NJ, USA). These experiments were performed in triplicate.
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
S.J. Lee et al. / Acta Biomaterialia xxx (2016) xxx–xxx
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Step I. Preparation of Scaffold for Bone Tissue Regeneration
Step II. In vitro test of pre-osteoblast cell
Fig. 1. Schematic illustration of hybrid 3D porous PCL scaffold and in vitro experiments.
2.5. Cytotoxicity of rhBMP2 and cell proliferation on 3D scaffolds The pre-osteoblast like cell line MC3T3-E1 was obtained from American Type Culture Collection (Manassas, VA). This cell line was used to perform the in vitro cell studies. In order to evaluate cytotoxicity, MC3T3-E1 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin in a 5% CO2 incubator at 37 °C. First, MC3T3-E1 cell were seeded onto 48well plates at a density of 5 104 cells per well. After 2 h, the cell culture medium was exchanged with varying concentrations of standard cell culture medium containing rhBMP2 (0, 1, 5, and 50 ng/ml). The cytotoxicity of rhBMP2 was evaluated at 24 and 48 h of incubation. For the in vitro cell proliferation study of the BMP2 loaded scaffolds, the MC3T3-E1 cells were drop seeded onto the scaffolds at a density of 5 104 cells per well in 50 ll of media. After 2 h, standard medium was filled on the scaffolds in each culture plate, respectively. Cell proliferation was determined at 1, 3, and 7 days. The cytotoxicity and cell proliferation were evaluated by using the cell counting kit (CCK-8) assay (n = 4). The absorbance of the medium was measured at 450 nm using a microplate reader (ELISA, Bio-Rad, Hercules, CA, USA). These experiments were repeated in triplicate.
2.6. ALP staining of rhBMP2 For ALP staining, MC3T3-E1 cells (a density of 5 104 cells per well) were seeded on each pristine well (n = 4, 48 well culture plate) using cell culture medium. After 2 h, medium was exchanged with osteogenic medium alone and varying quantities
of rhBMP2 (1, 10, and 50 ng/ml) in osteogenic medium were added. ALP staining was evaluated after incubation for 7 days and performed by using a kit purchased from Sigma–Aldrich (St. Louis, MO, USA). Briefly, the cell-seeded wells were fixed with 3.7% formaldehyde at room temperature for 30 min. After washing with DPBS carefully, the cells were incubated with a mixture of sodium nitrite solution, FRV-alkaline solution, and naphthol AS-BI alkaline solution according to the manufacturer’s protocol. After staining, the cell-seeded wells were washed twice with DPBS carefully and observed by optical microscopy (Olympus CKX41, JAPAN). These experiments were repeated in triplicate.
2.7. ALP activity and calcium deposition assay by alizarin red staining of scaffolds For ALP activity and alizarin red staining of scaffolds, the MC3T3-E1 cells were drop seeded onto scaffolds at a density of 5 104 cells per well using 50 ll of cell culture medium. After 2 h, the standard medium and osteogenic medium were filled with each well in the culture plate, respectively. The medium was changed every 3 days. The ALP activity was evaluated after 7 days and calcium deposition assay was examined at 14 day. For the ALP activity, attached cells on the scaffold were washed with DPBS twice and detached using trypsin. The harvested cells were then lysed using 1 RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40 and 1 mM EDTA) with a protease inhibitor cocktail (Boehringer Mannheim GmbH, Germany) for 30 min on an ice bath. Each of the lysates was centrifuged at 13,000 rpm at 4 °C for 15 min to remove the cell
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
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S.J. Lee et al. / Acta Biomaterialia xxx (2016) xxx–xxx Table 1 Base sequences of osteogenic primers for real time PCR. Primer name
Forward primer sequence (50 –30 )
Reverse primer sequence (50 –30 )
OCN COL 1 BSP GAPDH
GTC CAA GCA GGA GGG CAG CGT GGC GAC CAA GGT CCA GT CAG CGG CCC TGA GTC TGA CAA A CAT GGC CTT CCG TGT TCC TAC CC
TTG AGC TCA CAC ACC TCCC C AGG GAG ACC CAG AAT ACC GGG AG TCA CAA GCA GGG TTA AGC TCA CAC TG CCT CAG TGT AGC CCA AGA TGC CCT
debris. After centrifugation, the supernatant was collected and then reacted with p-nitrophenol phosphate solution (pNPP, Sigma) in a 5% CO2 humidified incubator at 37 °C for 30 min. The reaction with pNPP was then quenched by addition of 50 ll of 1 M NaOH. The quantity of p-nitrophenol was measured by absorption at 405 nm using a microplate reader. A calibration curve was generated using standard p-nitrophenol solutions. The quantity of produced total p-nitrophenol from cultured cells was obtained by comparing the absorption results against a calibration curve. The enzymatic activity was represented as lM of reaction product (p-nitrophenol) per minute per lg of total cellular protein. For the calcium deposition assay, the cell-seeded scaffolds were washed with DPBS twice, fixed for 20 min using 3.7% formaldehyde, and then washed with DPBS again. All the cells were stained in an incubator at 37 °C under 5% CO2 for 30 min using 40 mM alizarin red staining solution adjusted to pH 4.2. The staining solution was removed after 30 min, washed with distilled water several times carefully, and then was examined using an inverted fluorescence microscope. For quantitative analysis, the stained cells were desorbed with 10% 1-hexadecylpyridinium chloride and the absorbance was measured by using a microplate reader at 540 nm. These experiments were performed in triplicate. 2.8. Immunofluorescence of scaffolds for osteocalcin gene expression To confirm the gene expression of the cells under different conditions, immunostaining was performed. The MC3T3-E1 cells were drop seeded onto scaffolds comprised of PCLS, PCLSD, PCLSD 100, and PCLSD 500 at a density of 5 104 cells per well in 50 ll of media. After 2 h, standard medium and osteogenic medium were filled into each well in the culture plate, respectively. The immunochemistry was confirmed after 7 days. After PBS washing, the cell incubated scaffolds were fixed with 3.7% formaldehyde at 4 °C for 15 min. After being washed with PBS twice, the samples were washed with 0.5% Triton X-100 (in PBS) at room temperature for 10 min, and then blocked using 1% bovine serum albumin (in PBS) for 30 min at room temperature. After this, the samples were then incubated with the primary antibody against Osteocalcin (1:200 in 1% BSA; Thermo Scientific, Cat. #PA1-85754) for 1 h at room temperature, and then incubated by a rabbit anti-goat IgG secondary antibody Alexa FluorÒ 488 (1:200 in PBS; Invitrogen, Cat. #A-11078) for 1 h at room temperature following the standard procedure. The fluorescently labeled scaffolds were observed using confocal laser scanning microscopy (CLSM, Eclipse E600W, Nikon, Tokyo, Japan). The obtained images were assayed by using Nikon EZ-C1 software.
and afterward the entire RNA from each group’s cells was isolated using an RNeasy Plus Mini Kit (Qiagen, CA, USA) according to product manual. For each sample, 1 lg of the entire RNA was extracted and transcribed into cDNA using an AccuPower Cycle Script RT Premix (Bioneer, Daejeon, Republic of Korea). Real-time PCR was characterized by using iQ SYBR Green supermix (Bio-Rad, Hercules, CA, USA). Threshold cycle values were calculated by using a comparative cycle threshold (CT) method. The fold change of the control group (osteogenic medium only) after 7 days of incubation was set at 1-fold and the ratio of the normalized fold change was calculated. The RT-PCR amplifications were carried out for 30 s at 95 °C, 1 min at 58–62 °C and 1 min at 72 °C for 45 cycles after the initial denaturation step for 10 min at 95 °C. Each primer was designed for RT-PCR (Table 1) and purchased from Bioneer Co., Ltd. All the real time PCR results were normalized using a housekeeping gene consisting of a glyceraldehyde 3-phosphate dehydrogenase (GAPDH). These experiments were repeated in triplicate. 2.10. Analysis equipment The morphology of the scaffolds was observed using scanning electron microscopy (SEM, Hitachi S-4700, Japan) at an acceleration voltage of 15 kV. All samples were sputter-coated with platinum for 10 min. In order to estimate the amount of water uptake, the dried samples were initially weighed and subsequently immersed in a 10 ml vial with distilled water. At predetermined time points, the samples were weighed after removing the surface water using a Kim wipe. The water uptake (%) was calculated as follows:
Water uptakeð%Þ ¼ ½ðW after W before Þ=W before 100 Each sample’s contact angle was measured by using the water drop method using a video instrument (Phoenix 150, SEO, Korea). X-ray photoelectron spectroscopy (XPS) was performed using a K-Alpha 89 (Thermo Electron, UK) to confirm the surface chemistry. Alizarin red S stained scaffolds were visualized using a Smart G-scopeTM (Genie Technologies Inc., Philippines). 2.11. Statistical analysis Statistical analysis was performed using PASW Statistics 18 software (SPSS, Inc.). All values were expressed as means ± standard deviations, and differences with p-values (*p < 0.05) were considered statistically significant. 3. Results
2.9. Quantitative real-time polymerase chain reaction (RT-PCR)
3.1. Surface characterization of hybrid 3D scaffolds
For the RT-PCR experiment, the MC3T3-E1 cells were drop seeded onto scaffolds comprised of PCLS, PCLSD, PCLSD 100, and PCLSD 500 at a density of 5 104 cells per well in 50 ll of media. After 2 h, the standard medium and osteogenic medium were filled into each well in the culture plate, respectively. The RT-PCR assay was performed after 7 days of incubation. At each predetermined time interval, the cell-seeded wells were washed with DPBS twice
To confirm the morphology, the 3D-printed PCLS fabricated scaffold was observed by SEM analysis. Fig. 2a and b shows that these scaffolds possess an interlayered, layer-by-layer grid with approximately 400 lm strand and pore size. Subsequently, the PCLS was modified using DOPA chemistry. The results of this surface modification is represented in Fig. 3a and b. Fig. 3a shows the results of the water uptake experiment as performed against
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
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S.J. Lee et al. / Acta Biomaterialia xxx (2016) xxx–xxx
(b)
(a)
Fig. 2. Digital images (a) and SEM images (b) of fabricated 3D porous PCL scaffold, scale bar of full diagram is 500 lm and scale bar of small diagram is 750 lm.
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Time (sec) Fig. 3. Water uptake rate (a) and initial contact angle (a) of PCLS and PCLSD (n = 4, *p < 0.05 compared to PCLS groups).
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Fig. 4. XPS spectra (a) and surface chemical composition (b) of PCLS, PCLSD, PCLSD 100, PCLSD 500.
PCLS and PCLSD. Unmodified PCLS showed a very poor water uptake rate over the course of 60 s. However, the polydopaminecoated PCLS (PCLSD) was observed to rapidly absorb water. Additionally, the PCLSD was found to sink in cell-culture medium as shown in Fig. 3a. However, PCLS floated on the surface of the medium. These results were also confirmed via contact analysis. Fig. 3b shows that the bare PCLS has a water contact angle of 94° after 60 s. On the other hand, the PCLSD contact angle drops to near zero
point as soon as the water drop falls on it. This phenomenon was due to the hydrophilic DOPA. To verify the surface elemental chemical composition of the fabricated hybrid 3D scaffolds, XPS analysis was performed. The results are shown in Fig. 4a and b. In Fig. 4a and b, successful surface modification of the fabricated scaffold via DOPA was confirmed by unique amine group binding energy peaks (Fig. 4a). This was also confirmed by the increased N1s content (Fig. 4b). This is due to the introduction of amine
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
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S.J. Lee et al. / Acta Biomaterialia xxx (2016) xxx–xxx 200
Immobilized amount of rhBMP2 (ng)
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Fig. 5. Amount of attached rhBMP2 on PCLSD (a) and cumulative release profile of rhBMP2 from PCLSD 500 (b) (n = 4, *p < 0.05 compared to 100 ng/ml groups).
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Cell viability (%)
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TCP rhBMP2 (1ng/ml) rhBMP2 (10ng/ml) rhBMP2 (50ng/ml)
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Time (Hour) Fig. 6. Evaluation of MC3T3-E1 cells cytotoxicity toward TCP at various concentrations of rhBMP2 (1, 10, 50 ng/ml) in cell culture medium (a), ALP staining of MC3T3-E1 cells in osteogenic medium and rhBMP2 (1, 10, 50 ng/ml) loaded osteogenic medium (b), scale bar is 100 lm.
groups due to DOPA coating [23,27]. Furthermore, effective immobilization of rhBMP2 was verified by enhancement of N1s content as compared with the DOPA coated scaffold. PCLSD 500 was confirmed to have increased N1s content due to rhBMP2 relative to the PCLSD 100 indicating higher degree of rhBMP2 attachment (Fig. 4b). These results show that DOPA was well coated onto the PCLS and allowed for subsequent rhBMP2 attachment via facile surface treatment.
Our developed PCLSD 500 scaffolds exhibited a sustained release profile for up to 28 days with minimal burst release. This release tendency is consistent with previous reports which used DOPA chemistry to conjugate BMP2 to scaffolds [29]. Over the 28 day study, approximately 79.7% of rhBMP2 load was released from the PCLSD 500 scaffold.
3.3. In vitro evaluation of rhBMP2 effect on cells grown on the fabricated hybrid 3D scaffolds 3.2. Quantification of immobilized rhBMP2 and release kinetics of rhBMP2 from hybrid 3D scaffolds To verify the grafting efficiency of rhBMP2, the amount of unattached rhBMP2 was quantified by collecting the soaking solution immediately after completing the loading process. Fig. 5a shows the amount of immobilized rhBMP2 on DOPA coated scaffolds. This result shows that the amount of immobilized rhBMP2 was enhanced by increasing the concentration of rhBMP2 treatment. 22.7 ± 5 ng of rhBMP2 was immobilized on the surface when the scaffold was treated with 100 ng/ml of rhBMP2. For the scaffold treated with 500 ng/ml of rhBMP2, 153.3 ± 2.4 ng of rhBMP2 was immobilized on the surface. Based on these results, we anticipated that PCLSD 500 scaffold may induce a higher level of osteogenic activity as compared with the PCLSD 100 due to the higher concentration of rhBMP2 as rhBMP2 induces osteogenic differentiation [28]. We also performed a release test of rhBMP2 from the PCLSD 500. The rhBMP2 release profile of rhBMP2 is shown in Fig. 5b.
Cell cytotoxicity test was performed up to 48 h to ensure rhBMP2 did not present toxic effects at the concentrations delivered. Additionally, ALP staining after 7 days of incubation was performed in order to determine the effect of rhBMP2 on differentiation. In the present research, the MC3T3-E1 cells were employed for in vitro tests because they are pre-osteoblast cells which can differentiate into osseous tissue. As shown in Fig. 6a, cell viability at various concentration of rhBMP2 (1, 10, and 50 ng/ml) was retained above 100% as compared with bare tissue culture plate (TCP) indicating that there was no cell cytotoxicity for up to 48 h of incubation. In order to certify the osteogenic effect of rhBMP2 (1, 10, and 50 ng/ml), the ALP staining assay was examined after 7 days of incubation. The ALP stained MC3T3-E1 cells were visualized using an inverted fluorescence microscope as shown in Fig. 6b. There was no substantial color change between osteogenic medium and 1 ng/ml of rhBMP2. However, higher concentrations of rhBMP2 including 10 and 50 ng/ml resulted in a
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
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S.J. Lee et al. / Acta Biomaterialia xxx (2016) xxx–xxx
Cell proliferation (%)
400 PCLS PCLSD PCLSD 100 PCLSD 500
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* 200
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0 1
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Time (Day) Fig. 7. Cell proliferation of MC3T3-E1 cells grown on PCLS, PCLSD, PCLSD 100, and PCLSD 500 for 7 days (n = 4, *p < 0.05 compared to PCLS groups).
deep violet color as compared to bare medium and 1 ng/ml of rhBMP2. Interestingly, a significant color change was only observed with rhBMP2 at a concentration greater than 1 ng/ml indicating that this level is necessary for inducing osteogenic activity of MC3T3-E1 cells. For this reason, we anticipated that our developed scaffolds can play an effective role to promoting bone tissue formation because the surface treated scaffolds (PCLSD) contained a large amount of rhBMP2 on the surface (Fig. 5a). In order to determine the cell viability of the developed scaffolds (PCLS, PCLSD, PCLSD 100, and PCLSD 500), a cell proliferation test was performed using the CCK-8 assay. The results of this test are shown in Fig. 7. The viability of MC3T3-E1 cells on the scaffolds were compared relative to 1 day of incubation on PCLS as a control. The cell proliferation rate of all of groups increased after incubation for 7 days. Moreover, the DOPA coated PCLS (PCLSD) displayed a significantly different proliferation rate as compared with bare scaffolds (PCLS) for up to 7 days of incubation. The ALP activity after 7 days of incubation and alizarin red s assay after 14 days of incubation were examined in order to evaluate the bone differentiation activity of our developed scaffolds. As shown in Fig. 8a and b, the ALP activity results and the alizarin red s assay results were in good agreement with each other. Calcium deposition was also observed and this is shown in Fig. 8c (see white arrows in diagram). This also correlated to the results of the ALP activity and alizarin red s assay. The osteogenic effects also depended on whether non-osteogenic medium or osteogenic medium was used for incubation indicating that the differentiation medium can considerably affect cellular differentiation. Based on
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4. Discussion Bone tissue loss can be caused by many factors including bone degenerative disorders, physical trauma, unsuspected accidents, and large bone defects due to cancer. Because of the many causes of bone defect it is important to address this problem in order to increase the quality of healthcare worldwide [30,31]. In order to solve these issues, many surgeons have utilized autograft treatment for repairing bone defects which uses bone from a healthy region of the patient to fill the defect [32]. This method however has drawbacks in that the bone must be harvested from another region as well as the techniques involved are cumbersome [33]. To solve this issue, 3D bio-printing systems have been applied as a means to obviate the need for autograft surgical techniques [7,14]. Particularly, rapid prototyping techniques have played a very significant role in manufacturing bio-mimetic porous scaffolds that allow complex shapes for bone tissue engineering [15,34]. Therefore, this technique has been considered as a means to enable the fabrication of porous structures which mimic the original tissue matrix [14]. For this reason, we utilized a 3D printing system to fabricate bone regenerative scaffolds (Fig. 1). PCL is a semi-crystalline, linear, resorbable, aliphatic polyester approved by the U.S. Food and Drug Administration (FDA) for use in medical devices. This polymer has been investigated for use as a bone regenerative biological scaffold because of its biodegradable properties [35,36]. This polymer also has a very low melting point and glass transition temperature which enables 3D printing of this
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this comparison, we assessed our developed scaffolds to determine whether they improve differentiation of MC3T3-E1 cells. The level of ALP activity and amount of mineralized calcium were significantly increased by introducing rhBMP2 onto PCLSD. The osteogenic activity was found to be dose-dependent and considerably higher by grafting a larger amount of rhBMP2. These results indicate that our developed products can accelerate osteogenic differentiation of MC3T3-E1 cells by delivering rhBMP2. In order to confirm these results, we evaluated the mRNA expression levels of osteocalcin (OCN), collagen type 1 (Col1), and bone sialoprotein (BSP) through real-time PCR assay after 7 days of incubation. These results are shown in Fig. 9b–d. It was found that the rhBMP2 induced scaffolds (PCLSD 100 and PCLSD 500) substantially increased the mRNA expression levels for all markers as compared to the PCLS and PCLSD group. This result was supported by immunochemistry. This is shown visually in Fig. 9a. The OCN protein was stained green in order to aid visualization. The change in fluorescence due to rhBMP2 effect was clear. Furthermore, the PCLSD 500 had higher fluorescence as compared with PCLSD 100.
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Fig. 8. ALP activity after incubation for 7 days (a), amount of calcium deposition (b) and alizarin red s staining (c) after incubation of MC3T3-E1 cells on PCLS for 14 days, PCLSD, PCLSD 100, and PCLSD 500 (n = 4, *p < 0.05 compared to PCLS osteogenic medium groups, scale bar is 400 lm).
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
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Fig. 9. Immunostaining of osteocalcin gene (a) and real-time PCR analysis of osteocalcin (b), collagen type 1 (c), and Bone sialoprotein (d) of MC3T3-E1 cells grown on PCLS (non-osteogenic medium and osteogenic medium), PCLSD, PCLSD 100, and PCLSD 500 after incubation for 7 days (n = 4, *p < 0.05 compared to PCLS osteogenic medium groups, scale bar is 400 lm).
material to form a controlled porous scaffold [37]. Despite these advantages, PCL is a hydrophobic polymer which has no active sites for cellular attachment. These properties can hinder cell adhesion and migration [18,19]. Thus, we introduced hydrophilicity by coating the PCLS using DOPA chemistry in order to enhance the bone like-cell attachment and to promote cellular migration (Fig. 3). In the previous report, Jo et al. demonstrated that bioplotted 3D PCL scaffolds modified with DOPA chemistry had good cell infiltration into the scaffolds, which facilitates cell proliferation and migration [38]. Similarly, our cell viability results indicated that the PCLSD also showed good cell proliferation as compared with bare PCLS (Fig. 7). It is well known that surface wettability is an important factor for function of mammalian cells as well as osteoblast adhesion on biomaterials and that these parameters are crucial for bone tissue regeneration [39,40]. Due to the DOPA effect in current work, our cell viability results indicated that the PCLSD showed good cell proliferation as compared with bare PCLS. The DOPA-coated substrate can also act to conjugate to peptides and bioactive molecules containing primary amine and/or thiol groups via imine formation or Michael addition [24,27,41]. In the present study, we employed DOPA chemistry for the effective delivery of rhBMP2 with a sustained release pattern in order to promote bone tissue regeneration (Fig. 5). As mentioned in Section 1, the DOPA has catechol and amine groups within the dopamine. This allows for the polymer to form on any substrate with slightly alkaline pH [23]. This allows for great potential in bone
regeneration as well as mediated biomolecule dosing [29,42]. Thus, numerous studies have focused on the combination of DOPA and BMP for bone tissue engineering [43]. Despite its excellent performance, the exact mechanism of bioactive grafting of bioactive molecules on the DOPA layer is still controversial [29]. Further studies may be required in the future to elucidate this mechanism. In order to accelerate the osteogenic activity in this study, we used rhBMP2 to induce cellular differentiation for bone tissue regeneration. We chose rhBMP2 because bone morphogenetic proteins (BMPs) are multifunctional cytokines that belong to the transforming growth factor (TGF-b) superfamily of proteins and also play a crucial role in bone formation. BMPs are also approved by the FDA [44–46]. Moreover, BMPs are well known to mediate cell proliferation, differentiation, and to promote osteogenesis [28,47]. BMPs are also an essential signaling molecule involved in the recovery of fractured bone [26]. Previously, research by Wang’s group established that implantation of rhBMP2 can promote new bone formation in vivo [48,49]. Additionally, Lee et al. showed that DOPA-coated electrospun poly (lactide-co-glycolic acid) nanofibers can immobilize bone forming peptide-1 derived from bone morphogenetic protein-7 and serve to promote bone tissue regeneration [25]. Ko et al. demonstrated that porous poly (lactideco-glycolic acid) scaffolds can serve to conjugate osteoinductive biomolecules (recombinant human bone morphogenetic protein2; rhBMP2) via DOPA chemistry. These scaffolds possessed greater bone regeneration than bare scaffolds [50]. Additionally, Cho et al.
Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006
S.J. Lee et al. / Acta Biomaterialia xxx (2016) xxx–xxx
utilized BMP2 grafted poly(L-lactide) nanofibrous scaffold using DOPA chemistry for guided bone regeneration. They also investigated the effect of these scaffolds on bone formation and cell behavior [29]. In this study, we performed the cytotoxicity experiment of rhBMP2 as low concentration of 0, 1, 10, and 50 ng/ml to determine whether the bed effect against MC3T3-E1 cell prior to scaffolds test. Through this work, we clearly confirmed that rhBMP2 did not harmful influence for MC3T3-E1 cells viability. Based on these result, our developed outcomes have confirmed the possibility of bone tissue-engineered scaffolds because our fabricated scaffolds can immobilize the rhBMP2 at the quantity of 22.7 ± 5 ng when treatment with 100 ng/ml rhBMP2 and 153.3 ± 2.4 ng when treated with 500 ng/ml rhBMP2 in quantification of the grafting content of rhBMP2 experiment (Fig. 5a). Through this experiment, we verified that the immobilized rhBMP2 onto scaffold was greater than minimum amount of rhBMP2 regarding cell cytotoxicity test (allowable range: rhBMP2 (50 ng) > PCLSD 100 (22.7 ± 5 ng) > rhBMP2 (10 ng)). Based on this finding, we conducted the cell proliferation experiment on 3D scaffolds which also did not show cell cytotoxicity (Fig. 7). Prior to confirm the scaffold of an osteogenesis activity, we performed the cell proliferation test of rhBMP2 immobilized scaffolds using pre-osteoblast cell. In result, the DOPA coated PCLS (PCLSD) was close to that of group attached with rhBMP2 for up to 7 days of incubation (Fig. 7). As mentioned general knowledge, bone morphogenic protein can contribute cell proliferation and osteodifferentiation of osteoblast cell. However, our cell proliferation experiment revealed that rhBMP2 immobilized scaffolds did not show a significant increase of proliferation compared to that of developed scaffolds. There is shown a very slice increase. This result was consistent with our previous reports [51] which also shown a very slice increase using rhBMP2 agent for bone tissue regeneration. This report concluded that the effects of rhBMP-2 on osteoblast proliferation are still unclear. Further study, Lee et al. [52] showed that cell proliferation of BMP2 immobilized sample was higher compared to other groups, but there was no statistical significance. There results concluded that all substrates are safe and suitable materials for the growth of osteoblasts. Based on these opinions, we considered result of current our proliferation test was also no problem. Thus, we believe that our 3D printed scaffolds can provide not only a good environment for cell proliferation, but also an effective platform for dose-controlled delivery of rhBMP2 (Fig. 5b) for bone tissue regeneration. We also conducted the alp staining experiment of rhBMP2 as low concentration of 0, 1, 10, and 50 ng/ml to determine whether the bone differentiation against MC3T3-E1 cell prior to scaffolds test. As shown in Fig. 6b, a significant color change was only observed with rhBMP2 at a concentration greater than 1 ng/ml indicating that this level is necessary for inducing osteogenic activity of MC3T3-E1 cells. As previous mentioned, through quantification of the grafting content of rhBMP2 analysis, we verified that the immobilized rhBMP2 onto scaffold was greater than minimum amount of rhBMP2 regarding cell test (PCLSD 100 (22.7 ± 5 ng) > rhBMP2 (10 ng)). Based on this finding, our developed outcomes have confirmed the possibility of bone tissue differentiation scaffold. As an expectation, more amount of rhBMP2 may indicate the better promoting bone tissue formation. However, we didn’t feel the need for high concentrations of rhBMP2 study because bone differentiation of MC3T3-E1 cells was begun at 10 ng of rhBMP2 which confirmed the alp staining. As expected, our in vitro tests using ALP activity and mRNA gene expression indicated that these scaffolds induced osteogenic activity (Figs. 7–9). The ALP activity and three types of bonerelated gene expression such as OCN, Col1, and BSP were greater in PCLSD 500 as compared to the other groups. This result is due to rhBMP2 which was well grafted onto these scaffolds and serves to promote osteoinduction [26,53,54]. The above in vitro results
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suggest that our developed scaffolds exhibit improved osteogenic properties and may play a key role for bone tissue regeneration. 5. Conclusions In summary, the 3D porous scaffolds were successfully prepared by 3D printing system and these possessed the microporous architecture. Application of a DOPA coating significantly promoted the surface wettability of the PCL scaffolds as well as effectively allowed for grafting of rhBMP2 which was then released in a sustained manner. The scaffolds were tested in vitro and were found to exhibit good cellular activity for both cell proliferation and osteogenic activity. All of these results demonstrate that our strategy provides for synergistic effects including a good environment for cell proliferation and potent delivery of rhBMP2. This allowed for the generation of scaffolds which possessed good properties for use as bone-tissue scaffolds. Acknowledgments This study was supported by a grant from The Korean Health Technology R&D Project (HI13C1527), Ministry of Health & Welfare, Republic of Korea. References [1] S. Bose, M. Roy, A. Bandyopadhyay, Recent advances in bone tissue engineering scaffolds, Trends Biotechnol. 30 (2012) 546–554. [2] J.K. Park, J.H. Shim, K.S. Kang, J. Yeom, H.S. Jung, J.Y. Kim, et al., Solid free-form fabrication of tissue-engineering scaffolds with a poly (lactic-co-glycolic acid) grafted hyaluronic acid conjugate encapsulating an intact bone morphogenetic protein-2/poly (ethylene glycol) complex, Adv. Funct. Mater. 21 (2011) 2906– 2912. [3] J. Zeltinger, J.K. Sherwood, D.A. Graham, R. Müeller, L.G. Griffith, Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition, Tissue Eng. 7 (2001) 557–572. [4] V. Karageorgiou, D. Kaplan, Porosity of 3D biomaterial scaffolds and osteogenesis, Biomaterials 26 (2005) 5474–5491. [5] C.A. Custódio, R.L. Reis, J.F. Mano, Engineering biomolecular microenvironments for cell instructive biomaterials, Adv. Healthcare Mater. 3 (2014) 797–810. [6] C. Vitale-Brovarone, E. Verné, L. Robiglio, P. Appendino, F. Bassi, G. Martinasso, et al., Development of glass-ceramic scaffolds for bone tissue engineering: characterisation, proliferation of human osteoblasts and nodule formation, Acta Biomater. 3 (2007) 199–208. [7] C.R. Kothapalli, M.T. Shaw, M. Wei, Biodegradable HA-PLA 3-D porous scaffolds: effect of nano-sized filler content on scaffold properties, Acta Biomater. 1 (2005) 653–662. [8] H. Guo, J. Su, J. Wei, H. Kong, C. Liu, Biocompatibility and osteogenicity of degradable Ca-deficient hydroxyapatite scaffolds from calcium phosphate cement for bone tissue engineering, Acta Biomater. 5 (2009) 268–278. [9] J.B. Lee, S.I. Jeong, M.S. Bae, D.H. Yang, D.N. Heo, C.H. Kim, et al., Highly porous electrospun nanofibers enhanced by ultrasonication for improved cellular infiltration, Tissue Eng. Part A 17 (2011) 2695–2702. [10] X. Wu, Y. Liu, X. Li, P. Wen, Y. Zhang, Y. Long, et al., Preparation of aligned porous gelatin scaffolds by unidirectional freeze-drying method, Acta Biomater. 6 (2010) 1167–1177. [11] D.W. Hutmacher, Scaffolds in tissue engineering bone and cartilage, Biomaterials 21 (2000) 2529–2543. [12] S.V. Murphy, A. Atala, 3D bioprinting of tissues and organs, Nat. Biotechnol. 32 (2014) 773–785. [13] S.J. Hollister, Porous scaffold design for tissue engineering, Nat. Mater. 4 (2005) 518–524. [14] B. Derby, Printing and prototyping of tissues and scaffolds, Science 338 (2012) 921–926. [15] S. Bose, S. Vahabzadeh, A. Bandyopadhyay, Bone tissue engineering using 3D printing, Mater. Today 16 (2013) 496–504. [16] S.A. Park, S.H. Lee, W.D. Kim, Fabrication of porous polycaprolactone/ hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering, Bioprocess Biosyst. Eng. 34 (2011) 505–513. [17] S.A. Park, J.B. Lee, Y.E. Kim, J.E. Kim, J.H. Lee, J.-W. Shin, et al., Fabrication of biomimetic PCL scaffold using rapid prototyping for bone tissue engineering, Macromol. Res. 22 (2014) 882–887. [18] H.A. Declercq, T. Desmet, E.E. Berneel, P. Dubruel, M.J. Cornelissen, Synergistic effect of surface modification and scaffold design of bioplotted 3-D poly-ecaprolactone scaffolds in osteogenic tissue engineering, Acta Biomater. 9 (2013) 7699–7708.
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Please cite this article in press as: S.J. Lee et al., Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering, Acta Biomater. (2016), http://dx.doi.org/10.1016/j. actbio.2016.02.006