Journal of Biomaterials Applications http://jba.sagepub.com/
Poly(dopamine) coating to biodegradable polymers for bone tissue engineering Wei-Bor Tsai, Wen-Tung Chen, Hsiu-Wen Chien, Wei-Hsuan Kuo and Meng-Jiy Wang J Biomater Appl 2014 28: 837 DOI: 10.1177/0885328213483842 The online version of this article can be found at: http://jba.sagepub.com/content/28/6/837
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Article
Poly(dopamine) coating to biodegradable polymers for bone tissue engineering
Journal of Biomaterials Applications 2014, Vol 28(6) 837–848 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328213483842 jba.sagepub.com
Wei-Bor Tsai1, Wen-Tung Chen1, Hsiu-Wen Chien1, Wei-Hsuan Kuo2 and Meng-Jiy Wang2
Abstract In this study, a technique based on poly(dopamine) deposition to promote cell adhesion was investigated for the application in bone tissue engineering. The adhesion and proliferation of rat osteoblasts were evaluated on poly(dopamine)-coated biodegradable polymer films, such as polycaprolactone, poly(L-lactide) and poly(lactic-co-glycolic acid), which are commonly used biodegradable polymers in tissue engineering. Cell adhesion was significantly increased to a plateau by merely 15 s of dopamine incubation, 2.2–4.0-folds of increase compared to the corresponding untreated substrates. Cell proliferation was also greatly enhanced by poly(dopamine) deposition, indicated by shortened cell doubling time. Mineralization was also increased on the poly(dopamine)-deposited surfaces. The potential of poly(dopamine) deposition in bone tissue engineering is demonstrated in this study. Keywords Chondrocyte, osteoblasts, biodegradable polymers, dopamine, mineralization, cell attachment and proliferation
Introduction Scaffold, a critical element in tissue engineering, plays a role like the extracellular matrix (ECM) in natural tissues for supporting cell attachment, proliferation and differentiation. Synthetic degradable polymers, such as poly-L-lactic acid (PLLA), polyglycolic acid, their copolymers poly(L-co-glycolic acid) (PLGA) and polycaprolactone (PCL), are popularly used in fabrication of tissue engineering scaffolds due to their advantages of controllable degradation rates, high reproducibility, good processibility and the fact that they are approved for clinical use. Nevertheless, synthetic biodegradable polymers usually lack biological signals of the ECM, and thus may not be sufficient to support cell adhesion, growth and differentiated phenotypes. Several strategies have been applied to improve cell affinity of synthetic substrates. The most favored method mimics in vivo cell adhesion mechanisms to the ECM. For example, physical adsorption or chemical conjugation of ECM adhesion proteins or peptides to biomaterial surfaces enhances cell adhesion and proliferation via integrin–ligand interactions.1–5 Another strategy is to conjugate a pair of molecules that bind to each other, such as avidin–biotin, onto cells and biomaterial
surfaces to enhance the affinity between cells and substrates.6,7 Recently, a facile and versatile surface modification technique based on marine mussels’ adhesive mechanism provides a new tool to increase cell affinity.8 Marine mussels bind tightly to various surfaces on which they reside in aqueous environment, which relies on exhaustively repeated 3,4-dihydroxy-L-phenylalanine-lysine (DOPA-K) motif found in mussel adhesive proteins.9,10 Inspired by the structure of the DOPA-K motif, Messersmith’s group8 recently applied dopamine to create versatile poly(dopamine) (PDA) ad-layers on a wide range of organic and inorganic materials, such as noble metals, metals with native oxide surfaces, oxides, semiconductors, ceramics and synthetic polymers. Dipping substrates in an alkaline dopamine
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Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan 2 Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan Corresponding author: Wei-Bor Tsai, Department of Chemical Engineering, National Taiwan University, No. 1, Roosevelt Road, Section 4, Taipei 106, Taiwan. Email: [email protected]
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solution (e.g. pH 8.5) spontaneously create a thin adherent PDA film due to oxidative polymerization of dopamine.10 A PDA ad-layer on bioinert substrates is capable of promoting protein adsorption and cell adhesion.11–16 Furthermore, a PDA coating provides surface chemical reactivity for conjugation of a wide variety of molecules. For example, a PDA layer is able to react with thiols and amines via Michael addition or Schiff base reaction.13,17–20 This mussel-inspired surface functionalization strategy is extremely useful in biomaterial applications because the process does not require timeconsuming synthesis of complex linkers and is solventfree and non-toxic. Previously, Ku et al.12 showed that PDA surface promoted the adhesion and spreading of mouse osteoblast MC3T3-E1 cells to adhere to various non-wetting surfaces such as polyethylene, polytetrafluoroethylene, silicon rubber and polydimethylsiloxane that do not support cell adhesion well.12 Nevertheless, the growth and differentiation of osteoblasts, which is an important for bone tissue engineering, were not investigated in their study. In this study, this simple surface modification technique was applied to enhance the affinity of several biodegradable polymers to osteoblasts. PDA deposition was applied to several types of degradable polymer films at various deposition times to evaluate the attachment and proliferation of rat osteoblasts. Furthermore, the effect of PDA coating on mineralization of osteoblasts was investigated for long-term culture to evaluate the efficacy of PDA deposition on bone tissue engineering.
Preparation and characterization of samples Polymer films were cast on 96-well polypropylene plates (Corning, USA). PLGA was dissolved in N,Ndimethylformamide while PCL and PLLA were dissolved in chloroform to a concentration of 0.25% (w/ v). Each polymer solution was added onto polypropylene plates (100 mL/well) and allowed evaporation to form a thin film. Residual solvents in polymer films were further removed in a vacuum oven at 90 C for at least 12 h. 3-Hydroxytyramine hydrochloride (dopamine hydrochloride, cat. # H8502, Sigma-Aldrich) was dissolved in 10 mM Tris buffer (pH 8.5) to 1 mg/mL. All the polymer films were rinsed with deionized water before incubation with 100 mL dopamine solution at room temperature for various periods. After dopamine incubation, the samples were rinsed with deionized water. Surface hydrophobicity and elemental compositions of the PDA-deposited PCL films were characterized by static water contact angle measurement and electron spectroscopy for chemical analysis (ESCA), respectively. Static water contact angles of the samples were measured at seven locations on each type of degradable polymer films using a goniometer (FTA-125, First Ten A˚ngstroms, USA) with the sessile drop technique (2 -mL drop of deionized water). ESCA spectra for the samples were recorded on a VG Microtech MT-500 Spectrometer (UK) with an Mg Ka X-ray source radiation. The atomic compositions of the surfaces were calculated from the high-resolution spectrum of each element.
Materials and methods Materials
Osteoblast culture on PDA-coated polymer films
Reagents were received from Sigma-Aldrich (St. Louis, USA) unless specified otherwise. PCL (cat. # 440744, Mn ¼ 80,000) was obtained from Aldrich. PLGA (85/ 15, cat. # 85LG-300) and PLLA (cat. # LP-200) were purchased from Bioinvigor (Taipei, Taiwan). Tris(hydroxymethyl) aminomethane (Tris-base) was received from J. T. Baker (cat. # 4109-02). Osteoblast culture medium contained a-minimum essential medium (HyClone, USA), 10% fetal bovine serum (JRH Biosciences, Australia), 0.0679% (v/v) 2mercaptoethanol, 200 mg/mL gentamicin (GIBCOÕ , Invitrogen, USA) and 25 mg/mL fungizone (GIBCO), pH 7.4. The osteoblast culture medium supplemented with 1 mM sodium glycerophosphate, 0.1 mM dexamethasone and 50 mg/mL L-ascorbate constituted osteoblast differentiation medium. Phosphate buffered saline (PBS) contained 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 (pH 7.4), and was sterilized by an autoclave.
Standard sterile cell culture techniques were used for all cell experiments. The animal experiments followed the ethical guidelines of Care and Use of Laboratory Animals of National Taiwan University (National Institutes of Health Publication No. 85-23, revised, 1985), and the procedure was approved by the Animal Center Committee of National Taiwan University. Primary osteoblasts were isolated from neonatal rate calvariae. Briefly, Wistar rats at 1–3 days old were euthanized by decapitation and calvariae were then isolated. After the removal of periostea, the calvariae were cut into small pieces (1–2 mm2) and incubated in digestion solution (1 mg/mL Type I collagenase in PBS) at 37 C for 15 min. The digestion solution was then replaced by fresh digestion solution for 2 h of incubation at 37 C. Finally, the digestion solution was collected and centrifuged at 1000 rpm. The cell pellets were resuspended in the osteoblast culture medium. The identity of isolated osteoblasts was
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verified by alkaline phosphatase (ALP) staining according to a previous procedure.21 The number and viability of isolated osteoblasts were determined by using a hemocytometer with trypan blue staining. Rat osteoblasts at the second passage were used in this study. Prior to cell seeding, the polymer films were sterilized with 70% alcohol, rinsed with PBS three times and then immersed in the osteoblast culture medium for 1 h. Osteoblasts were seeded at 1 104 cells/cm2. Cell culture medium was changed every 3 days. Cell morphology was observed under a phase contrast microscope. Cell adhesion and growth to the polymer films were determined by a lactate dehydrogenase method, which was modified from a previous procedure22 and reported previously.23 Briefly, adhered cells were lysed by 0.5% (v/v) Triton X-100 (150 mL/well) for 30 min. Then, 50 mL of each lysate was mixed with an equal volume of a reaction solution (12 mg/mL sodium L-lactate, 1 mg/mL b-nicotinamide adenine dinucleotide, 0.9 mg/mL diaphorase, 0.1% (w/v) bovine serum albumin, 4 mg/mL sucrose and 0.067 mg/mL iodonitrotetrazolium chloride in PBS) and then incubated at 37 C for 20 min. The reaction was stopped by the addition of 50 mL oxamate solution (16 mg/mL sodium oxamate in PBS). The absorbance at 490 nm (OD490) was measured with a microtiter plate reader (model EL800, Bio-Tek, USA). The number of adherent cells was determined according to a calibration curve generated by plotting OD490 versus a series of osteoblasts suspensions with known cell numbers. A standard curve is shown in the Supplementary Material (Figure S1). Cell doubling time was calculated according to the following equation, Nt ¼ N0 2t=td , where Nt is the cell number after t days of culture, N0 the initial cell number and td the cell doubling time. Intracellular ALP activities were assayed by determining the release of p-nitrophenol from 4-nitrophenyl phosphate disodium salt.21 Briefly, 50 mL of cell lysate in 1% Triton was mixed with 100 mL 2-amino-2-methyl1 propanol buffer (0.5 M 2-amino-2-methyl-1 propanol, 8 mM 4-nitrophenyl phosphate disodium salt hexahydrate, 2 mM MgCl2, pH 10), and incubated in the dark at 37 C for 1 h. The reaction was stopped by adding 100 mL of 0.5 N NaOH, and the absorbance at 405 nm was read with a microtiter plate reader. A series of known concentrations of p-nitrophenol were used to generate a calibration curve for determining ALP activities.
Mineralization culture Osteoblasts were seeded on the substrates in the osteoblast culture medium for 7 days of culture. The culture medium was then replaced with the osteoblast differentiation medium. L-Ascorbate was replenished freshly
every day. After culturing for additional 14 days, the cells were fixed with 1% glutaraldehyde in PBS and rinsed with DI water. Alizarin red S solution (pH 5.5) was added and incubated at room temperature for 20 min, followed by rinses with DI water. The stained samples were observed under a light microscope, and the images were taken by using a digital camera. The total amount of calcium deposition was determined using a calcium assay kit (Diagnostic Chemicals Limited, USA) according to a previous protocol.24
Statistical analysis The data were reported as mean standard deviation. The statistical analyses between different groups were performed by using GraphPad Instat 3.0 (GraphPad Software, USA) with Student’s t-test. Probabilities of p 0.05 were considered as significant differences.
Results and discussion The attachment of osteoblasts on PDA-deposited substrates The impact of PDA deposition on osteoblast adhesion was first investigated with varied dopamine incubation time (5, 10, 15, 30 and 60 min). The morphology of attached osteoblasts was examined on the unmodified and dopamine-modified polymer films after 1 day of culture. Osteoblasts displayed spread morphology at different extents on the unmodified PCL, PLLA and PLGA, while PDA deposition further enhanced cell spreading regardless of the dopamine-coating time (Figure 1 for PLGA, more images in Figure 7 of the appendix). Furthermore, it is obvious that more cells are attached to the PDA-deposited surfaces compared with the unmodified ones, so the cell numbers were next quantified. The attachment of osteoblasts to the unmodified biodegradable polymers was varied in an order of PLGA > PLLA > PCL (Figure 2(a)). With 5-min dopamine deposition, cell adhesion was greatly enhanced on all the types of the substrates by 2.71 -, 1.84 - and 1.85-fold on PCL, PLGA and PLLA, respectively (p < 0.001 vs. unmodified substrates). Further increase in dopamine incubation time did not significantly increase cell adhesion compared to the substrates treated with 5-min dopamine incubation. Our previous study showed that the enhancement of chondrocyte adhesion to PDA-deposited surfaces was probably due to the increase in surface-immobilized serum fibronectin.11 In this study, the effect of serum proteins on cell adhesion to PDA-deposited PCL was evaluated by comparing cell attachment under the serum-containing and serum-free media. In the absence of serum, cell adhesion onto PCL was slightly increased
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Figure 2. One-day osteoblast adhesion to PCL deposited with PDA with different incubation time under serum-free or serumcontained medium. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD. ## p < 0.001 vs. PDA-deposited PCL.
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Figure 1. One-day osteoblast adhesion to PCL, PLGA and PLLA that were incubated with dopamine solution for 5 to 60 minutes. (a) microscopic images of cell adhesion to PDAdeposited PLGA. The time in each figure represents the incubation time in dopamine solution. Scale bar ¼ 100 mm. (b) Cell numbers on the PDA-deposited substrates after 1 day of cell culture. The solid circle represents cell adhesion to TCPS. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD.
after 10-min dopamine incubation (Figure 2(b)). Further increase in dopamine incubation time did not enhance cell adhesion. Nevertheless, osteoblast adhesion to PDA-deposited PCL was greatly enhanced under serum-containing condition (p < 0.001 vs. serum-free medium). Similar conclusion was also found on PLGA and PLLA (Figure 8 of the appendix). The results indicate that the attachment of osteoblasts onto PDA-deposited surfaces is mainly via serum adhesive proteins agglutinated on PDA substrates, but not due to cells that are directly ‘glued’ on PDA-deposited surfaces. Since cell adhesion to the PDA-deposited surfaces reached a plateau value on the surface coated with
Figure 3. Osteoblast numbers after 1 day of cell culture on the substrates incubated with dopamine solution for 15 s to 5 min. The solid circle represents cell adhesion to TCPS. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD.
dopamine for 5 min, we wondered what minimal dopamine incubation time can promote the adhesion of osteoblasts. Therefore, dopamine-coating time was reduced to evaluate the dependence of coating time on cell adhesion. Merely 15 s of dopamine coating increased cell adhesion to a plateau value, approximately four times of cell attachment on the unmodified PCL (Figure 3, p < 0.001). Similarly, 15-s dopamine deposition was sufficient to increase the adhesion of osteoblasts to a maximum on PLGA. On the other hand, cell adhesion to PLLA reached a plateau value after 4 min of dopamine deposition, although 15 s of dopamine deposition increased cell adhesion to 2.2fold compared with the unmodified PLLA (p < 0.001). Previously, we showed that dopamine deposition did not support full adhesion of chondrocytes until 3–4 min of incubation time.11 It seems that osteoblasts are more sensitive to PDA-deposited surfaces than chondrocytes.
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The results showed that only seconds of dopamine deposition are sufficient to significantly enhance cell adhesion in contrast to long period of dopamine incubation (16–24 h) in previous studies.12,25 Therefore, prolonged PDA deposition is not necessary. On the other hand, long PDA deposition time may not be desirable for tissue engineering applications. It is known that the thickness of a PDA ad-layer is increased with dopamine incubation time.8 The thickness of a PDA film was estimated to reach a value of 50 nm after 24 h of dopamine incubation.8 We previously tried to use proteases such as papain and trypsin to digest PDA but with success. It is questionable whether PDA is degradable in vivo. Since scaffolds are designed to be degraded and then replaced by the ECM secreted by cells, we suggest that it is better to deposit PDA film as thin as possible.
Surface characterization of PDA-deposited PCL The enhancement of cell adhesion by PDA deposition should be due to the alteration in surface properties. Surface modification of PCL via PDA deposition was investigated by static water contact angle measurements and ESCA measurement in this study. After PDA treatment, the static water contact angles of all the substrates were decreased with time (Table 1). After merely 1-min dopamine incubation, the static water contact angles were decreased by 2 , and the difference was further intensified with time. After 60 min of dopamine incubation, the static water contact angle was decreased to 85 . The ESCA measurement on the PCL substrates indicated that both oxygen and nitrogen contents were generally increased with the incubation time in dopamine solutions (Table 1). After 60 min of dopamine incubation, the contents of oxygen and nitrogen were much greater than the untreated PCL, indicating PDA deposition on the substrate. After 60 min of PDA deposition, the oxygen level was still less than the theoretical oxygen contents of PDA, while the nitrogen content was only half of the theoretical nitrogen value, suggesting that after 60-min PDA coating, the thickness of PDA deposition is less than the detectable depth of ESCA (around 10 nm). By comparing the data of surface characterization and cell attachment, it is noted that although surface modification of PCL by 15 s of PDA deposition was almost undetectable, cell adhesion was greatly increased. The only notable difference between surface elemental compositions after 15 s of PDA deposition is that the oxygen content was increased from 2.65% to 4.89%. Nevertheless, water contact angles were not varied. We could not explain why such slight surface modification was sufficient to support the attachment of osteoblasts.
Table 1. Static water contact angles and elemental compositions of PCL coated with poly(dopamine) for various coating time. Atomic composition (%) Dopamine Water contact incubation time angle ( ) C O N 0 15 s 30 s 1 min 5 min 10 min 15 min 30 min 60 min Polydopamine (theoretical)
96.37 3.08 95.95 2.14 93.22 3.02 94.15 4.95 93.32 1.02 91.77 2.07 92.11 1.51 89.32 2.06 84.88 3.55
96.64 94.59 95.28 88.57 89.78 84.72 83.79 83.88 79.54 72.72
2.65 4.89 4.18 10.33 8.61 13.79 13.85 12.76 16.2 18.18
0.71 0.52 0.54 1.10 1.61 1.49 2.36 3.36 4.26 9.09
The theoretic atomic composition of poly(dopamine) was calculated from the elemental composition of dopamine.
Osteoblast growth on PDA-coated polymer films We next evaluated whether PDA deposition enhances the growth of osteoblasts. PCL, PLGA and PLLA films were modified with 1-min PDA deposition. As shown in Figure 4, cell numbers on the PDA-modified surfaces were greatly enhanced during 5 days of culture. Compared with the unmodified PCL (2873 cells/cm2), the cell number on the PDA-deposited PCL was increased to 6944 cells/cm2 (2.41-fold) after 1 day of culture (p < 0.001). After 3 days of culture, the difference between the unmodified PCL and the PDA-deposited PCL increased to 6.37-fold (p < 0.001). After 5 days of culture, the cell numbers on the PDA-deposited PCL increased to 38,016 cells/cm2, much higher than that on the unmodified PCL (5891 cells/cm2, p < 0.001). The overall cell doubling time on the PDA-deposited PCL during 5 days of culture was 1.63 days, much less than the value on the unmodified PCL (3.86 days). Similarly, cell proliferation was enhanced by PDA deposition on PLGA and PLLA. The cell doubling times for the untreated and PDA-deposited PLGA were 4.87 days and 1.65 days, respectively, while the cell doubling times for the untreated and PDA-deposited PLLA were 2.65 days and 1.61 days, respectively. The cell doubling times on all the PDA-treated surfaces were comparable with the value on TCPS (1.88 days). The results indicated that PDA deposition greatly enhanced the proliferation of osteoblasts. The decrease in cell doubling time indicates that PDA deposition not only enhances the adhesion of osteoblasts but also the proliferation. Similar conclusion was found in our previous study regarding chondrocytes.11
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The increase in cell proliferation via PDA deposition might be due to an increase in serum adhesive proteins such as fibronectin, which enhances the interactions between cell integrins and artificial surfaces. Cell adhesion to the ECM adhesive proteins results in clustering of integrins, which is thought to be important in initiating integrin signals for cell growth.26 Furthermore, integrin binding to the ECM also leads to an organization of the cytoskeleton and cell spreading, which is important for cell proliferation.27,28
activities of osteoblasts on the PDA-deposited PCL. Osteoblasts were cultured on the PDA-deposited PCL for 7 days in osteoblast culture medium and then incubated in osteogenic culture medium for 14 days. During the normal culture condition, cell proliferation was greatly enhanced by PDA deposition (Figure 5(a)). The cell numbers on PCL modified with 1-min PDA deposition were less compared with the other PDAmodified PCL substrates (p < 0.01). No significant difference in cell numbers was found for the substrates deposited with PDA more than 5 min. ALP activity is an important indication for osteoblasts and is considered as a differentiation marker of osteoblastic phenotype.29 The cellular ALP activity was highest after 1 day of culture, decreased to a lowest level after 3 days of culture and then increased after 7 days of culture (Figure 5(b)). Nevertheless, the ALP activities remained a similar level on all PCL surfaces regardless of PDA deposition. The results indicate that PDA deposition does not enhance differentiated phenotype of osteoblasts. After 14 days of osteogenic culture, the samples were stained with alizarin red for the detection of calcium deposition. The images taken from a digital camera showed that compared with the untreated PCL, denser red stains were found on the PDA-treated PCL (Figure 6(a)). The quantified data of calcium deposition complied with the observation. The calcium deposition on the unmodified PCL (29 nmol/well) was much smaller compared with the DPA-modified PCL (p < 0.001, Figure 6(b)). The calcium deposition on the PCL with 1-min PDA deposition (314 nmol/sample) was less than the calcium content on the PCL with 5 min PDA deposition (518 nmol/sample; p < 0.001). Further increase in PDA-deposition time did not increase the calcium contents.
Mineralization of osteoblasts on dopamine-deposited PCL The mission of osteoblasts is to deposit mineralized matrix in a bone tissue. We investigated mineralization
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Figure 4. Osteoblasts culture on the substrates deposited with PDA for 1 min. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD. *p < 0.001 vs. the corresponding unmodified surfaces for the same culture period.
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Figure 5. Osteoblast culture on the PCL deposited with PDA for different incubation time: (a) cell numbers and (b) ALP activities. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD. *p < 0.001 vs. the corresponding unmodified PCL for the same culture period.
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(A)
several biodegradable polymers by soaking them in alkaline dopamine solution for only a short period of time from seconds to minutes. The increase in osteoblast adhesion should be due to the increased immobilization of serum adhesive proteins such as fibronectin on the PDA ad-layer. Mineralization was also increased on the PDA-deposited surfaces. The potential of PDA surface modification on bone tissue engineering in vivo studies needs future investigation.
Funding This study was financially supported by the National Science Council, Taiwan (grant no. 100-2221-E-002114-MY2).
Conflict of interest None declared. (B) 700
References
Calcium (nmol)
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Figure 6. Calcium deposition of osteoblasts cultured on the PCL deposited with PDA for different incubation time after 14 days of osteogenic culture. (a) The images of alizarin red-stained samples (96-well plate, 6.5 mm-in-diameter). The time in each figure represents the incubation time in dopamine solution. (b) The amount of calcium deposition by osteoblasts on different samples. n ¼ 5 and value ¼ mean SD. *p < 0.001 vs. the PDA-deposited PCL surfaces.
Although the ALP activity of osteoblasts is not enhanced by PDA deposition, mineralization was greatly enhanced on the PDA-deposited PCL. The difference suggests that the increase in calcium deposition via PDA deposition is not due to an elevation of differentiated phenotype of osteoblasts, but likely owing to more abundant cell population on the PDA-deposited PCL compared with the unmodified PCL.
Conclusion In the present work, we have shown that osteoblast adhesion and proliferation significantly increased to
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neural stem cell engineering. Biomaterials 2012; 33: 6952–6964. Tsai WB, Chen RP, Wei KL, et al. Polyelectrolyte multilayer films functionalized with peptides for promoting osteoblast functions. Acta Biomater 2009; 5: 3467–3477. Grunkemeier JM, Tsai WB, Alexander MR, et al. Platelet adhesion and procoagulant activity induced by contact with radiofrequency glow discharge polymers: roles of adsorbed fibrinogen and vWF. J Biomed Mater Res 2000; 51: 669–679. Chien HW, Wu SP, Kuo WH, et al. Modulation of hemocompatibility of polysulfone by polyelectrolyte multilayer films. Colloids Surf B Biointerf 2010; 77: 270–278. Chien HW, Tan SF, Wei KL, et al. Modulation of the functions of osteoblast-like cells on poly(allylamine hydrochloride) and poly(acrylic acid) multilayer films. Colloids Surf B Biointerf 2011; 88: 297–303. Ku SH and Park CB. Human endothelial cell growth on mussel-inspired nanofiber scaffold for vascular tissue engineering. Biomaterials 2010; 31: 9431–9437. Giancotti FG and Tarone G. Positional control of cell fate through joint integrin/receptor protein kinase signaling. Annu Rev Cell Dev Biol 2003; 19: 173–206. Chen CS, Mrksich M, Huang S, et al. Geometric control of cell life and death. Science 1997; 276: 1425–1428. Davey G, Buzzai M and Assoian RK. Reduced expression of alpha 5 beta B1 integrin prevents spreadingdependent cell proliferation. J Cell Sci 1999; 112: 4663–4672. Weinreb M, Shinar D and Rodan GA. Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization. J Bone Miner Res 1990; 5: 831–842.
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Appendix
Figure 7. Microscopic images of osteoblast adhesion to (A) PCL, (B) PLGA and (C) PLLA that were incubated with dopamine solution for various time after one day of culture. Scale bar = 100 m.
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Figure 7. Continued.
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Figure 7. Continued.
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Figure 8. One-day osteoblast adhesion to (a) PLGA and (b) PLLA deposited with PDA with different incubation time under serumfree or serum-contained medium. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD (# p < 0.01, ## p < 0.001 vs. unmodified surfaces in serum medium; *p < 0.001).
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Poly(dopamine) coating to biodegradable polymers for bone tissue engineering Wei-Bor Tsai, Wen-Tung Chen, Hsiu-Wen Chien, Wei-Hsuan Kuo and Meng-Jiy Wang J Biomater Appl 2014 28: 837 DOI: 10.1177/0885328213483842 The online version of this article can be found at: http://jba.sagepub.com/content/28/6/837
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Article
Poly(dopamine) coating to biodegradable polymers for bone tissue engineering
Journal of Biomaterials Applications 2014, Vol 28(6) 837–848 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0885328213483842 jba.sagepub.com
Wei-Bor Tsai1, Wen-Tung Chen1, Hsiu-Wen Chien1, Wei-Hsuan Kuo2 and Meng-Jiy Wang2
Abstract In this study, a technique based on poly(dopamine) deposition to promote cell adhesion was investigated for the application in bone tissue engineering. The adhesion and proliferation of rat osteoblasts were evaluated on poly(dopamine)-coated biodegradable polymer films, such as polycaprolactone, poly(L-lactide) and poly(lactic-co-glycolic acid), which are commonly used biodegradable polymers in tissue engineering. Cell adhesion was significantly increased to a plateau by merely 15 s of dopamine incubation, 2.2–4.0-folds of increase compared to the corresponding untreated substrates. Cell proliferation was also greatly enhanced by poly(dopamine) deposition, indicated by shortened cell doubling time. Mineralization was also increased on the poly(dopamine)-deposited surfaces. The potential of poly(dopamine) deposition in bone tissue engineering is demonstrated in this study. Keywords Chondrocyte, osteoblasts, biodegradable polymers, dopamine, mineralization, cell attachment and proliferation
Introduction Scaffold, a critical element in tissue engineering, plays a role like the extracellular matrix (ECM) in natural tissues for supporting cell attachment, proliferation and differentiation. Synthetic degradable polymers, such as poly-L-lactic acid (PLLA), polyglycolic acid, their copolymers poly(L-co-glycolic acid) (PLGA) and polycaprolactone (PCL), are popularly used in fabrication of tissue engineering scaffolds due to their advantages of controllable degradation rates, high reproducibility, good processibility and the fact that they are approved for clinical use. Nevertheless, synthetic biodegradable polymers usually lack biological signals of the ECM, and thus may not be sufficient to support cell adhesion, growth and differentiated phenotypes. Several strategies have been applied to improve cell affinity of synthetic substrates. The most favored method mimics in vivo cell adhesion mechanisms to the ECM. For example, physical adsorption or chemical conjugation of ECM adhesion proteins or peptides to biomaterial surfaces enhances cell adhesion and proliferation via integrin–ligand interactions.1–5 Another strategy is to conjugate a pair of molecules that bind to each other, such as avidin–biotin, onto cells and biomaterial
surfaces to enhance the affinity between cells and substrates.6,7 Recently, a facile and versatile surface modification technique based on marine mussels’ adhesive mechanism provides a new tool to increase cell affinity.8 Marine mussels bind tightly to various surfaces on which they reside in aqueous environment, which relies on exhaustively repeated 3,4-dihydroxy-L-phenylalanine-lysine (DOPA-K) motif found in mussel adhesive proteins.9,10 Inspired by the structure of the DOPA-K motif, Messersmith’s group8 recently applied dopamine to create versatile poly(dopamine) (PDA) ad-layers on a wide range of organic and inorganic materials, such as noble metals, metals with native oxide surfaces, oxides, semiconductors, ceramics and synthetic polymers. Dipping substrates in an alkaline dopamine
1
Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan 2 Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan Corresponding author: Wei-Bor Tsai, Department of Chemical Engineering, National Taiwan University, No. 1, Roosevelt Road, Section 4, Taipei 106, Taiwan. Email: [email protected]
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solution (e.g. pH 8.5) spontaneously create a thin adherent PDA film due to oxidative polymerization of dopamine.10 A PDA ad-layer on bioinert substrates is capable of promoting protein adsorption and cell adhesion.11–16 Furthermore, a PDA coating provides surface chemical reactivity for conjugation of a wide variety of molecules. For example, a PDA layer is able to react with thiols and amines via Michael addition or Schiff base reaction.13,17–20 This mussel-inspired surface functionalization strategy is extremely useful in biomaterial applications because the process does not require timeconsuming synthesis of complex linkers and is solventfree and non-toxic. Previously, Ku et al.12 showed that PDA surface promoted the adhesion and spreading of mouse osteoblast MC3T3-E1 cells to adhere to various non-wetting surfaces such as polyethylene, polytetrafluoroethylene, silicon rubber and polydimethylsiloxane that do not support cell adhesion well.12 Nevertheless, the growth and differentiation of osteoblasts, which is an important for bone tissue engineering, were not investigated in their study. In this study, this simple surface modification technique was applied to enhance the affinity of several biodegradable polymers to osteoblasts. PDA deposition was applied to several types of degradable polymer films at various deposition times to evaluate the attachment and proliferation of rat osteoblasts. Furthermore, the effect of PDA coating on mineralization of osteoblasts was investigated for long-term culture to evaluate the efficacy of PDA deposition on bone tissue engineering.
Preparation and characterization of samples Polymer films were cast on 96-well polypropylene plates (Corning, USA). PLGA was dissolved in N,Ndimethylformamide while PCL and PLLA were dissolved in chloroform to a concentration of 0.25% (w/ v). Each polymer solution was added onto polypropylene plates (100 mL/well) and allowed evaporation to form a thin film. Residual solvents in polymer films were further removed in a vacuum oven at 90 C for at least 12 h. 3-Hydroxytyramine hydrochloride (dopamine hydrochloride, cat. # H8502, Sigma-Aldrich) was dissolved in 10 mM Tris buffer (pH 8.5) to 1 mg/mL. All the polymer films were rinsed with deionized water before incubation with 100 mL dopamine solution at room temperature for various periods. After dopamine incubation, the samples were rinsed with deionized water. Surface hydrophobicity and elemental compositions of the PDA-deposited PCL films were characterized by static water contact angle measurement and electron spectroscopy for chemical analysis (ESCA), respectively. Static water contact angles of the samples were measured at seven locations on each type of degradable polymer films using a goniometer (FTA-125, First Ten A˚ngstroms, USA) with the sessile drop technique (2 -mL drop of deionized water). ESCA spectra for the samples were recorded on a VG Microtech MT-500 Spectrometer (UK) with an Mg Ka X-ray source radiation. The atomic compositions of the surfaces were calculated from the high-resolution spectrum of each element.
Materials and methods Materials
Osteoblast culture on PDA-coated polymer films
Reagents were received from Sigma-Aldrich (St. Louis, USA) unless specified otherwise. PCL (cat. # 440744, Mn ¼ 80,000) was obtained from Aldrich. PLGA (85/ 15, cat. # 85LG-300) and PLLA (cat. # LP-200) were purchased from Bioinvigor (Taipei, Taiwan). Tris(hydroxymethyl) aminomethane (Tris-base) was received from J. T. Baker (cat. # 4109-02). Osteoblast culture medium contained a-minimum essential medium (HyClone, USA), 10% fetal bovine serum (JRH Biosciences, Australia), 0.0679% (v/v) 2mercaptoethanol, 200 mg/mL gentamicin (GIBCOÕ , Invitrogen, USA) and 25 mg/mL fungizone (GIBCO), pH 7.4. The osteoblast culture medium supplemented with 1 mM sodium glycerophosphate, 0.1 mM dexamethasone and 50 mg/mL L-ascorbate constituted osteoblast differentiation medium. Phosphate buffered saline (PBS) contained 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 (pH 7.4), and was sterilized by an autoclave.
Standard sterile cell culture techniques were used for all cell experiments. The animal experiments followed the ethical guidelines of Care and Use of Laboratory Animals of National Taiwan University (National Institutes of Health Publication No. 85-23, revised, 1985), and the procedure was approved by the Animal Center Committee of National Taiwan University. Primary osteoblasts were isolated from neonatal rate calvariae. Briefly, Wistar rats at 1–3 days old were euthanized by decapitation and calvariae were then isolated. After the removal of periostea, the calvariae were cut into small pieces (1–2 mm2) and incubated in digestion solution (1 mg/mL Type I collagenase in PBS) at 37 C for 15 min. The digestion solution was then replaced by fresh digestion solution for 2 h of incubation at 37 C. Finally, the digestion solution was collected and centrifuged at 1000 rpm. The cell pellets were resuspended in the osteoblast culture medium. The identity of isolated osteoblasts was
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verified by alkaline phosphatase (ALP) staining according to a previous procedure.21 The number and viability of isolated osteoblasts were determined by using a hemocytometer with trypan blue staining. Rat osteoblasts at the second passage were used in this study. Prior to cell seeding, the polymer films were sterilized with 70% alcohol, rinsed with PBS three times and then immersed in the osteoblast culture medium for 1 h. Osteoblasts were seeded at 1 104 cells/cm2. Cell culture medium was changed every 3 days. Cell morphology was observed under a phase contrast microscope. Cell adhesion and growth to the polymer films were determined by a lactate dehydrogenase method, which was modified from a previous procedure22 and reported previously.23 Briefly, adhered cells were lysed by 0.5% (v/v) Triton X-100 (150 mL/well) for 30 min. Then, 50 mL of each lysate was mixed with an equal volume of a reaction solution (12 mg/mL sodium L-lactate, 1 mg/mL b-nicotinamide adenine dinucleotide, 0.9 mg/mL diaphorase, 0.1% (w/v) bovine serum albumin, 4 mg/mL sucrose and 0.067 mg/mL iodonitrotetrazolium chloride in PBS) and then incubated at 37 C for 20 min. The reaction was stopped by the addition of 50 mL oxamate solution (16 mg/mL sodium oxamate in PBS). The absorbance at 490 nm (OD490) was measured with a microtiter plate reader (model EL800, Bio-Tek, USA). The number of adherent cells was determined according to a calibration curve generated by plotting OD490 versus a series of osteoblasts suspensions with known cell numbers. A standard curve is shown in the Supplementary Material (Figure S1). Cell doubling time was calculated according to the following equation, Nt ¼ N0 2t=td , where Nt is the cell number after t days of culture, N0 the initial cell number and td the cell doubling time. Intracellular ALP activities were assayed by determining the release of p-nitrophenol from 4-nitrophenyl phosphate disodium salt.21 Briefly, 50 mL of cell lysate in 1% Triton was mixed with 100 mL 2-amino-2-methyl1 propanol buffer (0.5 M 2-amino-2-methyl-1 propanol, 8 mM 4-nitrophenyl phosphate disodium salt hexahydrate, 2 mM MgCl2, pH 10), and incubated in the dark at 37 C for 1 h. The reaction was stopped by adding 100 mL of 0.5 N NaOH, and the absorbance at 405 nm was read with a microtiter plate reader. A series of known concentrations of p-nitrophenol were used to generate a calibration curve for determining ALP activities.
Mineralization culture Osteoblasts were seeded on the substrates in the osteoblast culture medium for 7 days of culture. The culture medium was then replaced with the osteoblast differentiation medium. L-Ascorbate was replenished freshly
every day. After culturing for additional 14 days, the cells were fixed with 1% glutaraldehyde in PBS and rinsed with DI water. Alizarin red S solution (pH 5.5) was added and incubated at room temperature for 20 min, followed by rinses with DI water. The stained samples were observed under a light microscope, and the images were taken by using a digital camera. The total amount of calcium deposition was determined using a calcium assay kit (Diagnostic Chemicals Limited, USA) according to a previous protocol.24
Statistical analysis The data were reported as mean standard deviation. The statistical analyses between different groups were performed by using GraphPad Instat 3.0 (GraphPad Software, USA) with Student’s t-test. Probabilities of p 0.05 were considered as significant differences.
Results and discussion The attachment of osteoblasts on PDA-deposited substrates The impact of PDA deposition on osteoblast adhesion was first investigated with varied dopamine incubation time (5, 10, 15, 30 and 60 min). The morphology of attached osteoblasts was examined on the unmodified and dopamine-modified polymer films after 1 day of culture. Osteoblasts displayed spread morphology at different extents on the unmodified PCL, PLLA and PLGA, while PDA deposition further enhanced cell spreading regardless of the dopamine-coating time (Figure 1 for PLGA, more images in Figure 7 of the appendix). Furthermore, it is obvious that more cells are attached to the PDA-deposited surfaces compared with the unmodified ones, so the cell numbers were next quantified. The attachment of osteoblasts to the unmodified biodegradable polymers was varied in an order of PLGA > PLLA > PCL (Figure 2(a)). With 5-min dopamine deposition, cell adhesion was greatly enhanced on all the types of the substrates by 2.71 -, 1.84 - and 1.85-fold on PCL, PLGA and PLLA, respectively (p < 0.001 vs. unmodified substrates). Further increase in dopamine incubation time did not significantly increase cell adhesion compared to the substrates treated with 5-min dopamine incubation. Our previous study showed that the enhancement of chondrocyte adhesion to PDA-deposited surfaces was probably due to the increase in surface-immobilized serum fibronectin.11 In this study, the effect of serum proteins on cell adhesion to PDA-deposited PCL was evaluated by comparing cell attachment under the serum-containing and serum-free media. In the absence of serum, cell adhesion onto PCL was slightly increased
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Figure 1. One-day osteoblast adhesion to PCL, PLGA and PLLA that were incubated with dopamine solution for 5 to 60 minutes. (a) microscopic images of cell adhesion to PDAdeposited PLGA. The time in each figure represents the incubation time in dopamine solution. Scale bar ¼ 100 mm. (b) Cell numbers on the PDA-deposited substrates after 1 day of cell culture. The solid circle represents cell adhesion to TCPS. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD.
after 10-min dopamine incubation (Figure 2(b)). Further increase in dopamine incubation time did not enhance cell adhesion. Nevertheless, osteoblast adhesion to PDA-deposited PCL was greatly enhanced under serum-containing condition (p < 0.001 vs. serum-free medium). Similar conclusion was also found on PLGA and PLLA (Figure 8 of the appendix). The results indicate that the attachment of osteoblasts onto PDA-deposited surfaces is mainly via serum adhesive proteins agglutinated on PDA substrates, but not due to cells that are directly ‘glued’ on PDA-deposited surfaces. Since cell adhesion to the PDA-deposited surfaces reached a plateau value on the surface coated with
Figure 3. Osteoblast numbers after 1 day of cell culture on the substrates incubated with dopamine solution for 15 s to 5 min. The solid circle represents cell adhesion to TCPS. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD.
dopamine for 5 min, we wondered what minimal dopamine incubation time can promote the adhesion of osteoblasts. Therefore, dopamine-coating time was reduced to evaluate the dependence of coating time on cell adhesion. Merely 15 s of dopamine coating increased cell adhesion to a plateau value, approximately four times of cell attachment on the unmodified PCL (Figure 3, p < 0.001). Similarly, 15-s dopamine deposition was sufficient to increase the adhesion of osteoblasts to a maximum on PLGA. On the other hand, cell adhesion to PLLA reached a plateau value after 4 min of dopamine deposition, although 15 s of dopamine deposition increased cell adhesion to 2.2fold compared with the unmodified PLLA (p < 0.001). Previously, we showed that dopamine deposition did not support full adhesion of chondrocytes until 3–4 min of incubation time.11 It seems that osteoblasts are more sensitive to PDA-deposited surfaces than chondrocytes.
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The results showed that only seconds of dopamine deposition are sufficient to significantly enhance cell adhesion in contrast to long period of dopamine incubation (16–24 h) in previous studies.12,25 Therefore, prolonged PDA deposition is not necessary. On the other hand, long PDA deposition time may not be desirable for tissue engineering applications. It is known that the thickness of a PDA ad-layer is increased with dopamine incubation time.8 The thickness of a PDA film was estimated to reach a value of 50 nm after 24 h of dopamine incubation.8 We previously tried to use proteases such as papain and trypsin to digest PDA but with success. It is questionable whether PDA is degradable in vivo. Since scaffolds are designed to be degraded and then replaced by the ECM secreted by cells, we suggest that it is better to deposit PDA film as thin as possible.
Surface characterization of PDA-deposited PCL The enhancement of cell adhesion by PDA deposition should be due to the alteration in surface properties. Surface modification of PCL via PDA deposition was investigated by static water contact angle measurements and ESCA measurement in this study. After PDA treatment, the static water contact angles of all the substrates were decreased with time (Table 1). After merely 1-min dopamine incubation, the static water contact angles were decreased by 2 , and the difference was further intensified with time. After 60 min of dopamine incubation, the static water contact angle was decreased to 85 . The ESCA measurement on the PCL substrates indicated that both oxygen and nitrogen contents were generally increased with the incubation time in dopamine solutions (Table 1). After 60 min of dopamine incubation, the contents of oxygen and nitrogen were much greater than the untreated PCL, indicating PDA deposition on the substrate. After 60 min of PDA deposition, the oxygen level was still less than the theoretical oxygen contents of PDA, while the nitrogen content was only half of the theoretical nitrogen value, suggesting that after 60-min PDA coating, the thickness of PDA deposition is less than the detectable depth of ESCA (around 10 nm). By comparing the data of surface characterization and cell attachment, it is noted that although surface modification of PCL by 15 s of PDA deposition was almost undetectable, cell adhesion was greatly increased. The only notable difference between surface elemental compositions after 15 s of PDA deposition is that the oxygen content was increased from 2.65% to 4.89%. Nevertheless, water contact angles were not varied. We could not explain why such slight surface modification was sufficient to support the attachment of osteoblasts.
Table 1. Static water contact angles and elemental compositions of PCL coated with poly(dopamine) for various coating time. Atomic composition (%) Dopamine Water contact incubation time angle ( ) C O N 0 15 s 30 s 1 min 5 min 10 min 15 min 30 min 60 min Polydopamine (theoretical)
96.37 3.08 95.95 2.14 93.22 3.02 94.15 4.95 93.32 1.02 91.77 2.07 92.11 1.51 89.32 2.06 84.88 3.55
96.64 94.59 95.28 88.57 89.78 84.72 83.79 83.88 79.54 72.72
2.65 4.89 4.18 10.33 8.61 13.79 13.85 12.76 16.2 18.18
0.71 0.52 0.54 1.10 1.61 1.49 2.36 3.36 4.26 9.09
The theoretic atomic composition of poly(dopamine) was calculated from the elemental composition of dopamine.
Osteoblast growth on PDA-coated polymer films We next evaluated whether PDA deposition enhances the growth of osteoblasts. PCL, PLGA and PLLA films were modified with 1-min PDA deposition. As shown in Figure 4, cell numbers on the PDA-modified surfaces were greatly enhanced during 5 days of culture. Compared with the unmodified PCL (2873 cells/cm2), the cell number on the PDA-deposited PCL was increased to 6944 cells/cm2 (2.41-fold) after 1 day of culture (p < 0.001). After 3 days of culture, the difference between the unmodified PCL and the PDA-deposited PCL increased to 6.37-fold (p < 0.001). After 5 days of culture, the cell numbers on the PDA-deposited PCL increased to 38,016 cells/cm2, much higher than that on the unmodified PCL (5891 cells/cm2, p < 0.001). The overall cell doubling time on the PDA-deposited PCL during 5 days of culture was 1.63 days, much less than the value on the unmodified PCL (3.86 days). Similarly, cell proliferation was enhanced by PDA deposition on PLGA and PLLA. The cell doubling times for the untreated and PDA-deposited PLGA were 4.87 days and 1.65 days, respectively, while the cell doubling times for the untreated and PDA-deposited PLLA were 2.65 days and 1.61 days, respectively. The cell doubling times on all the PDA-treated surfaces were comparable with the value on TCPS (1.88 days). The results indicated that PDA deposition greatly enhanced the proliferation of osteoblasts. The decrease in cell doubling time indicates that PDA deposition not only enhances the adhesion of osteoblasts but also the proliferation. Similar conclusion was found in our previous study regarding chondrocytes.11
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The increase in cell proliferation via PDA deposition might be due to an increase in serum adhesive proteins such as fibronectin, which enhances the interactions between cell integrins and artificial surfaces. Cell adhesion to the ECM adhesive proteins results in clustering of integrins, which is thought to be important in initiating integrin signals for cell growth.26 Furthermore, integrin binding to the ECM also leads to an organization of the cytoskeleton and cell spreading, which is important for cell proliferation.27,28
activities of osteoblasts on the PDA-deposited PCL. Osteoblasts were cultured on the PDA-deposited PCL for 7 days in osteoblast culture medium and then incubated in osteogenic culture medium for 14 days. During the normal culture condition, cell proliferation was greatly enhanced by PDA deposition (Figure 5(a)). The cell numbers on PCL modified with 1-min PDA deposition were less compared with the other PDAmodified PCL substrates (p < 0.01). No significant difference in cell numbers was found for the substrates deposited with PDA more than 5 min. ALP activity is an important indication for osteoblasts and is considered as a differentiation marker of osteoblastic phenotype.29 The cellular ALP activity was highest after 1 day of culture, decreased to a lowest level after 3 days of culture and then increased after 7 days of culture (Figure 5(b)). Nevertheless, the ALP activities remained a similar level on all PCL surfaces regardless of PDA deposition. The results indicate that PDA deposition does not enhance differentiated phenotype of osteoblasts. After 14 days of osteogenic culture, the samples were stained with alizarin red for the detection of calcium deposition. The images taken from a digital camera showed that compared with the untreated PCL, denser red stains were found on the PDA-treated PCL (Figure 6(a)). The quantified data of calcium deposition complied with the observation. The calcium deposition on the unmodified PCL (29 nmol/well) was much smaller compared with the DPA-modified PCL (p < 0.001, Figure 6(b)). The calcium deposition on the PCL with 1-min PDA deposition (314 nmol/sample) was less than the calcium content on the PCL with 5 min PDA deposition (518 nmol/sample; p < 0.001). Further increase in PDA-deposition time did not increase the calcium contents.
Mineralization of osteoblasts on dopamine-deposited PCL The mission of osteoblasts is to deposit mineralized matrix in a bone tissue. We investigated mineralization
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(A)
several biodegradable polymers by soaking them in alkaline dopamine solution for only a short period of time from seconds to minutes. The increase in osteoblast adhesion should be due to the increased immobilization of serum adhesive proteins such as fibronectin on the PDA ad-layer. Mineralization was also increased on the PDA-deposited surfaces. The potential of PDA surface modification on bone tissue engineering in vivo studies needs future investigation.
Funding This study was financially supported by the National Science Council, Taiwan (grant no. 100-2221-E-002114-MY2).
Conflict of interest None declared. (B) 700
References
Calcium (nmol)
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Figure 6. Calcium deposition of osteoblasts cultured on the PCL deposited with PDA for different incubation time after 14 days of osteogenic culture. (a) The images of alizarin red-stained samples (96-well plate, 6.5 mm-in-diameter). The time in each figure represents the incubation time in dopamine solution. (b) The amount of calcium deposition by osteoblasts on different samples. n ¼ 5 and value ¼ mean SD. *p < 0.001 vs. the PDA-deposited PCL surfaces.
Although the ALP activity of osteoblasts is not enhanced by PDA deposition, mineralization was greatly enhanced on the PDA-deposited PCL. The difference suggests that the increase in calcium deposition via PDA deposition is not due to an elevation of differentiated phenotype of osteoblasts, but likely owing to more abundant cell population on the PDA-deposited PCL compared with the unmodified PCL.
Conclusion In the present work, we have shown that osteoblast adhesion and proliferation significantly increased to
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Appendix
Figure 7. Microscopic images of osteoblast adhesion to (A) PCL, (B) PLGA and (C) PLLA that were incubated with dopamine solution for various time after one day of culture. Scale bar = 100 m.
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Figure 7. Continued.
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Figure 7. Continued.
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(b) serum-free medium serum medium ## * ## * *
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Figure 8. One-day osteoblast adhesion to (a) PLGA and (b) PLLA deposited with PDA with different incubation time under serumfree or serum-contained medium. Cell seeding density was 1 104 cells/cm2. n ¼ 5 and value ¼ mean SD (# p < 0.01, ## p < 0.001 vs. unmodified surfaces in serum medium; *p < 0.001).
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