Polydopamine-Assisted Osteoinductive Peptide Immobilization of

Aug 13, 2013 - Biomacromolecules , 2013, 14 (9), pp 3202–3213 ... Typical methods used for polymeric scaffold modification (e.g., ... Abstract | Ful...
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Polydopamine-Assisted Osteoinductive Peptide Immobilization of Polymer Scaffolds for Enhanced Bone Regeneration by Human Adipose-Derived Stem Cells Eunkyung Ko, Kisuk Yang, Jisoo Shin, and Seung-Woo Cho Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm4008343 • Publication Date (Web): 13 Aug 2013 Downloaded from http://pubs.acs.org on August 23, 2013

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Polydopamine-Assisted Osteoinductive Peptide Immobilization of Polymer Scaffolds for Enhanced Bone Regeneration by Human AdiposeDerived Stem Cells Eunkyung Ko1, Kisuk Yang1, Jisoo Shin1, Seung-Woo Cho1*

1

Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea

*Corresponding author Prof. Seung-Woo Cho Department of Biotechnology, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120749, Republic of Korea Tel: +82-2-2123-5662; Fax: +82-2-362-7265; E-mail: [email protected]

KEYWORDS: Polydopamine, Bone morphogenetic protein-2, Adipose-derived stem cell, Osteogenic differentiation, Bone regeneration

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ABSTRACT

Immobilization of osteoinductive molecules, including growth factors or peptides, on polymer scaffolds is critical for improving stem cell-mediated bone tissue engineering. Such molecules provide osteogenesis-stimulating signals for stem cells. Typical methods used for polymeric scaffold modification (e.g., chemical conjugation or physical adsorption), however, have limitations (e.g., multistep, complicated procedures, material denaturation, batch-tobatch inconsistency, and inadequate conjugation) that diminish the overall efficiency of the process. Therefore, in this study we report a biologically inspired strategy to prepare functional polymer scaffolds that efficiently regulate the osteogenic differentiation of human adipose-derived stem cells (hADSCs). Polymerization of dopamine (DA), a repeated motif observed in mussel adhesive protein, under alkaline pH conditions, allows for coating of a polydopamine (pDA) layer onto polymer scaffolds. Our study demonstrates that predeposition of a pDA layer facilitates highly efficient, simple immobilization of peptides derived from osteogenic growth factor (bone morphogenetic protein-2; BMP-2) on poly(lactic-co-glycolic acid) (PLGA) scaffolds via catechol chemistry. The BMP-2 peptideimmobilized PLGA scaffolds greatly enhanced in vitro osteogenic differentiation and calcium mineralization of hADSCs using either osteogenic medium or non-osteogenic medium. Furthermore, transplantation of hADSCs using pDA-BMP-2-PLGA scaffolds significantly promoted in vivo bone formation in critical-sized calvarial bone defects. Therefore, pDA-mediated catechol functionalization would be a simple and effective method for developing tissue engineering scaffolds exhibiting enhanced osteoinductivity. To the best of our knowledge, this is the first study demonstrating that pDA-mediated surface modification of polymer scaffolds potentiates the regenerative capacity of human stem cells for healing tissue defect in vivo. 2 ACS Paragon Plus Environment

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INTRODUCTION

Bone tissue engineering is a promising therapeutic technique for healing bone defects caused by traumatic bone injury, tumor resection, and osteitis. Several types of stem cells have been used as alternative cell sources for bone tissue engineering to replace primary osteogenic cells (e.g., osteoblasts) isolated from bone tissue biopsy.1, 2 In particular, adiposederived stem cells (ADSCs) have been highlighted for bone tissue engineering because they are easily harvested and cultured and are easily made to differentiate into the osteogenic lineage.3-7 The use of these stem cells induces greater bone regeneration than does transplantation of bone matrix alone into bone defects.8, 9 Several types of three-dimensional (3D) synthetic polymer scaffolds have been tested to enhance the osteogenic potential of transplanted ADSCs for facilitating bone formation.10-13 However, typical synthetic polymeric scaffolds lacking osteoinductive signals may not be effective in inducing bone regeneration following ADSC transplantation. The scaffolds therefore should be engineered to provide biochemical cues for promoting stem cell-mediated osteogenesis as well as proper physical and mechanical microenvironments. Modification of synthetic polymer scaffolds with osteoinductive growth factors or other molecules can compensate for the lack of osteogenic potential of the scaffolds. Several typical methods such as covalent chemical conjugation and physical adsorption have been used to immobilize growth factor proteins and peptides onto the surfaces of polymer scaffolds.14-19 Unfortunately, chemical conjugation methods usually require multistep, complicated procedures such as surface activation or other means of activation via pretreatment of oxygen plasma,14 irradiation,19 or chemicals.15-18 Because such treatments can significantly alter the bulk properties of materials, this may cause surface denaturation and batch-to-batch inconsistency.20, 21 In addition, chemicals used for surface activation and 3 ACS Paragon Plus Environment

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immobilization such as N-hydroxysuccinimide and maleimide are usually prone to hydrolysis, which leads to poor conjugation of proteins or peptides on the polymer surface.20, 21 Physical adsorption, another common method for immobilizing growth factor proteins and peptides, is usually non-specific and inefficient.20, 21 Therefore, simple, reliable, and efficient strategies to immobilize osteogenic factors would be of great importance for the development of highly osteoinductive polymer scaffolds for enhancing ADSC-mediated bone regeneration. A mussel-inspired immobilization strategy may provide an easy and efficient method to deposit proteins and peptides onto the polymeric scaffolds. Mytilus edulis foot protein-5 found in mussel adhesive pads contains an extensive repeat of 3,4-dihydroxy-Lphenylalanine (L-DOPA; dopamine (DA) precursor) residues, an attribute of which is to provide strong adhesion of the mussel to virtually any type of material surface (polymer, metal, glass, etc.).22, 23 DA contains a catechol functional group that promotes polymerization in alkaline conditions − a typical pH of marine environments − via oxidative conversion of catechol to quinone.22, 24 Polymerized DA, polydopamine (pDA), can be coated onto the material substrates in a thin layer.20-22 Diverse nucleophiles, including amine, thiol, or imidazole groups, can be covalently conjugated to catechol groups in a pDA layer. pDA coatings exhibit a latent affinity for various bioactive molecules containing those functional groups.20-22 Thus, a pDA-assisted surface modification might provide a process by which a wide variety of surfaces made of natural or synthetic organic/inorganic materials would be bonded with bioactive molecules.20-22 Previous studies reported that pDA-coated material substrates can be efficiently grafted with thiolated peptides,20 enzymes,21 polysaccharides,22 anti-cancer drugs,25 antibodies,26 or growth factor proteins (e.g., neurotrophic and angiogenic factors).20, 27, 28 Despite the capacity of pDA to efficiently immobilize bioactive molecules with diverse nucleophiles, pDA-mediated functionalization of polymer scaffolds to

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manipulate the therapeutic efficacy of stem cells still remains unexplored, especially in in vivo models with tissue defects. In this study, we report pDA-mediated immobilization of bone morphogenetic factor2 (BMP-2)-derived peptides onto biodegradable polymer [poly(lactic-co-glycolic acid), PLGA] scaffolds for enhancing the osteogenic potential of human ADSCs (hADSCs) in vitro and in vivo. BMP-2 signaling is essential for bone formation in the early stage of healing after bone fracture29-31 and also enhances the differentiation of ADSCs into the osteogenic lineage.32-34 Herein, osteoinductive peptides derived from BMP-2 sequence were efficiently immobilized onto PLGA scaffolds by a single step of pDA-mediated coating. Osteogenic differentiation of hADSCs was examined on pDA-BMP-2-PLGA substrates in vitro. Finally, PLGA scaffolds engineered with pDA-BMP-2 peptides were used for hADSC transplantation in a mouse model of critical-sized calvarial bone defect, and bone regeneration in the defects was evaluated by micro-computed tomography (micro-CT) and histological analysis 8 weeks after transplantation. pDA-assisted immobilization of BMP-2 peptides significantly enhanced osteogenic differentiation of hADSCs both in vitro and in vivo, leading to bone regeneration in the defects.

EXPERIMENTAL SECTION

pDA Coating. pDA coating was performed as previously described.20 PLGA films for in vitro culture and PLGA scaffolds for in vivo transplantation were coated with a pDA layer for subsequent immobilization of BMP-2 peptides. The films and scaffolds were immersed in a DA solution (2 mg/ml in 10 mM Tris-HCl, pH 8.5, Sigma, St. Louis, MO, USA) and placed on a shaker for 4 hours at room temperature. The pDA-coated films and scaffolds were then rinsed with distilled water to remove unattached DA molecules. 5 ACS Paragon Plus Environment

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Immobilization of BMP-2 Peptides. Peptide immobilization onto pDA-coated polymer substrates was performed as previously described.20 BMP-2 peptides (sequence: KIPKASSVPTELSAISTLYLGGK35) were immobilized onto pDA-coated PLGA films or scaffolds. The pDA-coated PLGA films and scaffolds were immersed in BMP-2 peptide solution (1 mg/ml in 10 mM Tris-HCl, pH 8.5, Peptron, Daejeon, Korea) for 6 hours at room temperature. The films and scaffolds were then washed with distilled water to remove the unattached peptides. For physical adsorption of BMP-2 peptides, PLGA films without pDA coating were immersed in BMP-2 peptide solution at the same concentration (1 mg/ml in 10 mM Tris-HCl, pH 8.5) and incubation conditions (6 hours at room temperature).

Determination of Peptide Immobilization Efficiency. The coating efficiency of BMP-2 peptides was determined by quantifying the amount of unattached peptides in the buffer solution retrieved immediately after completing the coating process. The peptide concentration was measured by using a fluorescamine assay (Sigma). In brief, after incubation of BMP-2 peptides onto pDA-coated PLGA substrates, the supernatants containing unattached peptides were retrieved and used as samples to determine the peptide immobilization efficiency. Fluorescamine assay was carried out by mixing fluorescamine solution (3 mg/ml in dimethyl sulfoxide) and the sample at 1:3 ratio in a 96-black well plate. After 15 minutes of incubation at room temperature, the fluorescence intensity of each sample was measured by using a microplate reader (GloMax-Multi Detection System, Promega, Madison, WI, USA). Finally, the amount of unattached BMP-2 peptides in the solution was calculated from the standard curve of BMP-2 peptides and subtracted from the amount of loaded peptides to quantify the amount of immobilized peptides. Immobilization efficiency was represented as the percentage ratio of the amount of immobilized peptides to 6 ACS Paragon Plus Environment

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the amount of loaded peptides (D0). The detachment of immobilized peptides was quantified by measuring the amount of released peptides from the engineered substrates during incubation under a physiologically relevant condition (in phosphate buffer saline (PBS) at 37°C). To this end, fresh PBS solution was added to BMP-2 peptide-immobilized PLGA substrates immediately after peptide immobilization and the substrates were then incubated at 37°C until the 6th day while taking the supernatants every day. The remaining peptides on the surface were quantified by subtracting the released peptides from the total amount of peptides immobilized on the substrates at the first day (D1~D6). The peptide immobilization efficiency of physical adsorption group (BMP-2-PLGA) was also determined with the same procedure.

Surface Characterization of Engineered PLGA Substrates. The atomic chemical composition and surface roughness of the engineered PLGA substrates were analyzed by Xray photoelectron spectroscopy (XPS) (K-Alpha, Thermo VG Scientific, East Grinstead, UK) and atomic force microscopy (AFM) (XE-100, Park Systems, Suwon, Korea), respectively. The water contact angle was measured by using a contact angle analyzer (Phoenix 300, SEO Corporation, Gunpo, Korea). The surface morphology of the engineered PLGA scaffolds was observed by scanning electron microscopy (SEM) (JSM 7001F, JEOL Ltd., Tokyo, Japan). Immobilization of peptides on the polymer films was visualized by using fluorescently labeled peptides (FITC-CGGRGD, Peptron). The fluorescent signals from immobilized FITC-CGGRGD peptides on the polymer films were detected by an image analyzer (Image Station 4000 MM, Kodak, New Haven, CT, USA).

Culture of Human Adipose-Derived Stem Cells (hADSCs). hADSCs were purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA) and cultured in Dulbecco’s Modified Eagle’s 7 ACS Paragon Plus Environment

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Medium (DMEM, Gibco, Gaithersburg, MD, USA) containing 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco) in humidified air with 5% CO2 at 37°C. The culture medium was exchanged every three days. The cells at passages 5-7 were used for differentiation experiments.

Osteogenic Differentiation of hADSCs on Engineered PLGA Substrates. hADSCs were seeded on pDA-BMP-2-PLGA films (diameter: 15 mm) at a seeding density of 4.0 × 104 cells/ml. Osteogenic differentiation of hADSCs was induced under culture conditions with either growth medium [DMEM supplemented with 10% (v/v) FBS] or osteogenic medium. Osteogenic medium was prepared by adding 10% (v/v) FBS, 0.1 µM dexamethasone (Sigma), 50 µM ascorbic acid (Sigma), and 10 mM β-glycerol phosphate (Sigma) in DMEM medium. Bare PLGA and pDA-coated PLGA films were tested as control substrate groups. For differentiation using osteogenic medium, the cells were first plated in growth medium and the medium was then changed to osteogenic medium 3 days after plating. The osteogenic differentiation medium was exchanged every three days.

Alizarin Red S Staining. After 21 days of culture, calcium deposition by hADSCs was analyzed by Alizarin Red S staining. The cells were fixed in 2.5% (v/v) glutaraldehyde (Sigma) in PBS for 15 minutes at room temperature and then washed with acidic PBS (pH 4.2) twice. The cells were then incubated in 2% (w/v) Alizarin Red S solution at 37°C for 20 minutes. The solution was freshly prepared before use by dissolving Alizarin Red S (Sigma) in deionized distilled water. After the removal of Alizarin Red S solution, the stained cells were rinsed with acidic PBS twice and observed under a light microscope (Olympus CKX41SF, Olympus, Tokyo, Japan).

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Immunocytochemistry. hADSCs cultured on PLGA films were fixed with 4% (w/v) paraformaldehyde (Sigma) and permeabilized with 0.1% (v/v) Triton X-100 (Sigma) for 15 minutes and 5 minutes, respectively. The cells were treated with 2% (v/v) goat blocking serum (Sigma) for 30 minutes to prevent non-specific binding of primary antibodies and then incubated with primary antibodies at 4°C overnight. The following primary antibodies were used for double immunofluorescent staining: mouse monoclonal anti-osteopontin (OPN) (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit polyclonal anti-collagen type I (COL I) (1:50; Calbiochem, San Diego, CA, USA). The cells were then incubated with fluorescent dye-conjugated secondary antibodies (Alexa Fluor-488 goat anti-mouse IgG and Alexa Fluor-594 donkey anti-rabbit IgG; Invitrogen) for 45 minutes at room temperature. Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). The fluorescent signals were observed under a fluorescent microscope (Olympus IX 71, Olympus, Tokyo, Japan).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). qRT-PCR was conducted after 21 days of culture, as previously reported.36 Total RNA was extracted from hADSCs cultured on the PLGA films (n=3 per group) by using RNeasy Mini kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer’s protocol. The concentration of RNA in each sample was determined by measuring absorbance at 260 nm using a spectrophotometer (Nanodrop ND-1000, Thermo Scientific, Waltham, MA, USA). The reverse transcription reaction to prepare cDNA from each RNA sample was performed by using Takara PrimeScript II First Strand cDNA Synthesis Kit (Takara, Shiga, Japan). qRTPCR was conducted by StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using Fast Universal PCR Master Mix (Applied Biosystems). The expression of osteogenic markers was quantified by TaqMan Gene Expression assays (Applied 9 ACS Paragon Plus Environment

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Biosystems) designed for each target (human OPN: Hs00959010_m1, human RUNX2: Hs00231692_m1, and human COL I: Hs00164004_m1). The expression level of each target gene was determined by using the comparative Ct method, normalizing expression to that of an endogenous reference transcript (human glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Hs02758991_g1).

Transplantation of hADSC-Seeded PLGA Scaffolds into a Mouse Model of Calvarial Bone Defect. We fabricated 3D microporous PLGA scaffolds (diameter: 4 mm, height: 1 mm, pore size: 100-300 µm) using a conventional solvent-casting and particulate leaching method.37 Undifferentiated hADSCs were seeded onto bare PLGA scaffolds, pDA-coated PLGA scaffolds, or pDA-BMP-2-immobilized PLGA scaffolds at a density of 1.0 × 106 cells/scaffold (n=8 per each group). The cells on PLGA scaffolds were cultured in growth medium without induction of osteogenic differentiation for 3 days after seeding and then transplanted into a mouse model of critical-sized calvarial bone defect. The seeded cells onto the polymer scaffolds usually need a certain period of time (3~5 days) to ensure cell adhesion before transplantation.37 All animal experiments were performed in accordance with the Korean Food and Drug Administration (KFDA) guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei Laboratory Animal Research Center (YLARC) (permit number: 2010-0049). All mice were maintained in the specific pathogen-free facility of the YLARC. The procedures for bone defect generation and transplantation are as follows.38 In brief, female athymic mice (Balb/cnu, 8 weeks old, Nara Bio Corporation, Pyungtaek, Korea) were anesthetized with xylazine (20 mg/kg, Bayer Korea, Ansan, Korea) and ketamine (100 mg/kg, Yuhan, Seoul, Korea). After incision of skin, calvarial bone defects (diameter: 4 mm) were made at two sites of the skull using a micro bone drill (Strong 102L, Saeshin, Daegu, Korea). The PLGA scaffolds 10 ACS Paragon Plus Environment

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with seeded hADSCs were put into the defects and the skin was then sutured by 6-0 sutures (Ethicon, Somerville, NJ, USA). The mice were divided into five groups, (i) no treatment, (ii) PLGA scaffold without hADSCs, (iii) PLGA scaffold with hADSCs, (iv) pDA-coated PLGA scaffold with hADSCs, and (v) pDA-BMP-2-immobilized PLGA scaffold with hADSCs. The mice were followed for 8 weeks and bone formation in the defect was analyzed.

Micro-Computed Tomography (Micro-CT). The calvarial bones were obtained 8 weeks after transplantation and fixed in 4% paraformaldehyde solution. Bone formation in the defected site was visualized by micro-CT systems (SkyScan-1172, SkyScan, Kontich, Belgium) and SkyScan CT analyzer program (SkyScan). The area of bone regeneration in the defect was quantified by using Image J software (National Institutes of Health, Bethesda, MD, USA).

Histology and Immunohistochemistry. The retrieved bone samples were fixed in 4% paraformaldehyde and then incubated in Decalcifying Solution-Lite (Sigma, 20:1 ratio (v/v) of Decalcifying Solution-Lite to tissue sample) at room temperature for 6 hours. Then, the samples were dehydrated with a series of ethanol and finally, embedded in paraffin. The tissues were sliced into 5-µm sections and the sections were stained using Goldner’s Trichrome method for detection of collagen in bone tissue. The nucleus was stained with hematoxylin, and the cytoplasm was subsequently stained with Goldner’s Trichrome Solution A. The tissue sections were washed three times with Goldner’s Trichrome Solution B, ionized with Goldner’s Trichrome Solution C, and then washed with Solution B again. Lastly, collagen was stained green with Goldner’s Trichrome Solution D. The tissue sections were serially dehydrated with ethanol and xylene. Newly formed collagen in the defect was observed under a light microscope. Collagen regeneration in the defected site was quantified 11 ACS Paragon Plus Environment

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as a percentage ratio of collagen-stained area to total defect area by using Image J software (National Institutes of Health). Tissue sections were also immunofluorescently stained with primary OPN-specific (1:100; Santa Cruz Biotechnology) and COL I-specific (1:50; Calbiochem) antibodies. The signals for each marker were visualized with Alexa Fluor 488and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen), respectively. The tissue sections were then observed under a fluorescent microscope (Olympus IX 71).

Statistical Analysis. Statistical analyses were conducted by a Student’s t test using SigmaPlot software (Systat Software Inc., Chicago, IL, USA). The data from quantitative analyses are presented as average ± standard deviation. Values of p0.05) in the difference between these two groups. In a previous study, pDA coating without further modification was found to promote osteogenic differentiation of human mesenchymal stem cells on the polymer scaffolds.48 Therefore, pDA coating alone may also have promoted commitment of hADSCs to osteogenic lineage in our study. The engineered PLGA substrates were also tested for osteogenic differentiation of hADSCs under non-osteogenic medium conditions. Although pDA-assisted BMP-2 immobilization enhanced osteogenic differentiation of hADSCs in the experiments using the medium supplemented with osteogenic factors, the presence of soluble factors complicates the assessment of the sole effect of pDA and BMP-2 peptides on hADSC differentiation. Therefore, the differentiation experiment was further conducted under non-osteogenic culture 18 ACS Paragon Plus Environment

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conditions to determine more clearly the effects of pDA and BMP-2 peptide coating on osteogenic differentiation of hADSCs. Calcium deposition after 21 days of culture was much less extensive in all of the PLGA substrate groups in non-osteogenic medium than in osteogenic medium condition because of the absence of osteogenic soluble factors (Figure 7A). Alizarin Red S staining revealed that calcium deposition was not detected in hADSCs cultured on the PLGA and pDA-PLGA substrate groups whereas BMP-2 immobilization induced calcium deposition of hADSCs (Figure 7A). Immunofluorescent staining of osteogenic markers (OPN and COL I) indicated a noticeable difference in osteogenic marker expression between pDA-BMP-2-PLGA substrate group and the bare PLGA or pDA-PLGA substrate group (Figure 7B). qRT-PCR analysis also demonstrated that pDA-mediated BMP2 peptide immobilization increased the expression of RUNX2, OPN, and COL I in hADSCs cultured on polymer substrate (Figure 7C-E). The difference in osteogenic marker expression between pDA-PLGA and pDA-BMP-2-PLGA groups was relatively small in osteogenic medium condition (Figure 6C-E) compared to non-osteogenic medium condition (Figure 7CE) due probably to the presence of osteogenic soluble factors including ascorbic acid, βglycerol phosphate, and dexamethasone. This may demonstrate the importance of medium choice in the experiments to assess the sole effect of biomaterials on stem cell differentiation.

In Vivo Bone Formation of hADSCs Using pDA-BMP-2 Peptide-Immobilized Scaffolds. Lastly, PLGA scaffolds engineered by pDA-mediated BMP-2 peptide immobilization were utilized to transplant hADSCs for bone regeneration in a mouse model of critical-sized calvarial bone defect. To check whether immobilized BMP-2 peptides can induce in vivo osteogenic differentiation of hADSCs, undifferentiated hADSCs were used for seeding, and the cells on the pDA-BMP-2-PLGA scaffolds were maintained in non-osteogenic medium for 3 days, prior to transplantation. Eight weeks after transplantation, bone formation in the 19 ACS Paragon Plus Environment

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defects was evaluated by micro-CT (Figure 8) and histological analysis (Figure 9 and 10). As confirmed by micro-CT, the no-treatment control and bare PLGA groups did not exhibit great amounts of calcium deposition or mineralized tissue formation (Figure 8A). In contrast, transplantation of hADSCs using the engineered PLGA scaffolds (pDA-PLGA and pDABMP-2-PLGA) enhanced mineralized tissue formation in the defects, compared with that observed with bare PLGA scaffolds with or without hADSCs (Figure 8A). Immobilization of BMP-2 peptides significantly reduced the unhealed area in the defect by facilitating mineralization process following hADSC transplantation (Figure 8B). Osteoinductive materials such hydroxyapatite (HA) were not used in this study. Because HA can be introduced onto various material surfaces including polymer scaffolds via pDA-mediated coating as reported by Ryu et al.,49 combined immobilization of HA and BMP-2 peptides on polymer scaffolds might further improve bone regeneration by hADSC transplantation. Histological analysis of the retrieved samples eight weeks after hADSC transplantation demonstrated the beneficial effect of pDA-mediated polymer scaffold modification with BMP-2 peptides on bone formation in the defects. Goldner’s Trichrome staining indicated scar tissue formation in the defect of control group (no treatment) (Figure 9A). Transplantation of PLGA scaffolds with or without hADSCs induced collagen formation in the defects, but the pDA-PLGA scaffold group with immobilized BMP-2 peptides exhibited the greatest collagen formation in the defected sites after hADSC transplantation (Figure 9A and B). New blood vessels with red blood cells were formed and observed to perfuse the host vasculature (red arrows in Figure 9A) in the groups receiving hADSC transplantation (PLGA-hADSC, pDA-PLGA-hADSC, and pDA-BMP-2-PLGAhADSC groups), probably arising from paracrine secretion of angiogenic growth factors from transplanted hADSCs.50, 51 This suggests the regeneration of vascularized bone tissue by hADSC transplantation using engineered PLGA scaffolds. Vascularization is critical for bone 20 ACS Paragon Plus Environment

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regeneration and persistence of newly formed bone tissue mass.52 Double immunofluorescent staining of osteogenic markers (OPN and COL I) indicated enhanced osteogenesis by hADSC transplantation using pDA-BMP-2-PLGA scaffolds (Figure 10). No treatment resulted in low expression of OPN and COL I in the defected site whereas application of pDA-PLGA or pDA-BMP-2-PLGA scaffold induced relatively greater expression of OPN and COL I (Figure 10), suggesting more extensive bone regeneration following hADSC transplantation. These results demonstrate that PLGA scaffolds engineered by pDA-mediated BMP-2 peptide coating can enhance osteogenic differentiation of hADSCs in vivo as well as in in vitro culture.

CONCLUSIONS

Our study suggests that mussel-inspired surface modification can provide a highly osteoinductive polymer scaffold capable of promoting osteogenic differentiation and mineralization of hADSCs in vitro and in vivo. pDA-mediated catechol functionalization allows for efficient, reliable immobilization of osteoinductive synthetic BMP-2 peptides onto polymer scaffolds. Transplantation of hADSCs using these osteoinductive polymer scaffolds significantly improved bone regeneration in the critical-sized bone defects. pDA-assisted surface modification can provide a useful platform technology for introducing other types of bioactive molecules that regulate stem cell self-renewal, proliferation, and differentiation for improved therapeutic efficacy in tissue engineering.

AUTHOR INFORMATION

Corresponding Author 21 ACS Paragon Plus Environment

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*E-mail: [email protected] (S.-W.C.)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work was supported by grants (2010-0022037 and 2010-0025982) funded by the National Research Foundation of Korea (NRF). This work was also supported by a grant (2009-0083522) from Translational Research Center for Protein Function Control (TRCP) funded by the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea. We thank Jin Kim for her assistance in preparing the schematic illustration in Figure 1.

REFERENCES

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Figure 1. Schematic illustration of pDA-assisted immobilization of osteoinductive BMP-2 peptides on 3D microporous PLGA scaffolds for enhancing osteogenic differentiation and bone formation of hADSCs.

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Figure 2. X-ray photoelectron spectroscopy (XPS) analysis of the engineered PLGA substrates. (A) XPS peaks of bare PLGA, pDA-PLGA, pDA-BMP-2-PLGA (prepared by pDA-mediated immobilization of BMP-2 peptides), and BMP-2-PLGA (prepared by physical adsorption of BMP-2 peptides) films. (B) Quantification of atomic chemical composition of each PLGA film surface.

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Figure 3. Efficiency of pDA-mediated peptide immobilization. (A) Fluorescamine assays were undertaken to determine the attachment efficiency of BMP-2 peptides onto pDA-coated or non-coated PLGA films in physiologically relevant conditions (in PBS at 37°C) for 6 days. (B) Visualization of fluorescently labeled peptides (FITC-CGGRGD and FITC-BMP-2) immobilized onto polymer substrates (PLGA, ePTFE, and PUA) with or without pDA coating.

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Figure 4. The surface roughness and wetting of the engineered PLGA substrates. (A) AFM images of bare PLGA film, pDA-coated PLGA film, and pDA-BMP-2 peptide-immobilized PLGA film. The scale bar indicates 10 µm. (B) Average surface roughness (Ra) of the PLGA films. (C, D) Water contact angles of bare PLGA film, pDA-coated PLGA film, and pDABMP-2 peptide-immobilized PLGA film (**; p