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Biological and Medical Applications of Materials and Interfaces
Cell-sheet-derived ECM coatings and their effects on BMSCs responses Xiaozhao Wang, Zun Chen, Beibei Zhou, Xiyue Duan, Wen-Jian Weng, Kui Cheng, Huiming Wang, and Jun Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19718 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018
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ACS Applied Materials & Interfaces
Cell-sheet-derived ECM coatings and their effects on BMSCs responses Xiaozhao Wang †, Zun Chen †, ǁ, Beibei Zhou †, Xiyue Duan †, Wenjian Weng †, Kui Cheng †, *, Huiming Wang ‡, #, Jun Lin #, * † School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou, 310027, China ǁ School of Medicine, Zhejiang University, Hangzhou, 3100058, China ‡ The Affiliated Stomatologic Hospital, Zhejiang University, Hangzhou, 310003, China # The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou, 310003, China ABSTRACT: Extracellular matrix (ECM) provides a dynamic and complex environment to determine the fate of stem cells. In this work, in vitro cultured cell sheets were treated with paraformaldehyde or ethanol and eventually become ECM. Such ECM was then immobilized on titanium substrates via polydopamine chemistry. Their effects on bone marrow mesenchymal stromal cells (BMSCs) behaviors were investigated. It was found that paraformaldehyde treated ECM coatings (PT-ECM) showed well-maintained microstructure, whereas that of ethanol treated (ET-ECM) were completely changed. As a result, different amide structures and distributions of ECM components, such as laminin and collagen I, were exhibited. Alkaline phosphatase activity, osteocalcin secretion, related gene expression and mineral deposition were evaluated for BMSCs cultured on both ECM coatings. PT-ECM was demonstrated to promote osteogenic differentiation much more efficiently than ET-ECM. That is ascribed to the preservation of native ECM milieu of PT-ECM. Such ECM acquirement and immobilization method could establish surfaces being able to direct stem cell responses on various materials. That shows promising potential in bone tissue engineering and other related biomedical applications. KEYWORDS: ECM coatings, cell sheets, polydopamine, BMSCs, osteogenic differentiation 1. INTRODUCTION Mesenchymal stem cells (MSCs) are one of the most widely available sources of stem cells, and thus possess great potential in cell-based therapy and regenerative medicines.1-2 Nevertheless, randomly differentiated stem cells are useless and even harmful in clinical.3 Thus, constructing a proper condition to direct the differentiation of MSCs is of great significance. Naturally, stem cells reside in a complex and active microenvironment consisting of extracellular matrix (ECM), which is replete with informational cues to direct cell behaviors.4 ECM not only serves as the structural support for stem cells but also stimulates various important biological functions, and eventually determines stem cell fate.5-6 Especially, the special niche of ECM could guide differentiation of MSCs into specific lineages. Extensive efforts have been made in designing biomaterials with ECM-mimicking structures, or ECM protein-containing
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biomaterials with laminin, collagen, fibronectin and etc.7-8 However, it’s still a dilemma to reproduce the function of the in vivo ECM. As such, several studies have set out to develop cellderived ECM by decellularization.9-12 Park et al revealed that different cell-derived ECM provided cells with distinct microenvironment, fibroblast-derived matrix and preosteoblastderived matrix showed a completely distinctive milieu.13 Moreover, ECM also makes contribution when it is immobilized on the surface of biomedical implants. Surface modification with cell-derived matrices shows great potential in reproducing the milieu of natural ECM.14-18 E.g., Harvestine et al demonstrated that ECM-coated composite scaffolds promote persistence and osteogenesis of MSCs.19 It is noteworthy that such property of ECM could be quite important for metallic bone implants, such as titanium (Ti), since their metallic surfaces are biologically inert and fails to fulfil the osteogenesis requirements. Thus, constructing a proper extracellular microenvironment on implant surface via surface modification to direct MSCs behaviors, especially osteogenic differentiation, is crucial to bone repairing materials. In recent years, cell sheet harvesting technology, allowing the easy in vitro development of thin-layer tissue, was intensively studied and utilized to construct three-dimensional tissues for further clinical applications in tissue engineering.20-21 One of the most significant reasons is the complete preservation of secreted ECM in the detached cell sheets.22 In addition, the culturing process of cell sheet makes it possible to obtain autologous ECM, which will reduce the possibility of immunologic rejection. Thus, in vitro harvested cell sheet could be an alternative source for easy acquisition of ECM. In this work, on the basis of previously developed light-induced-cell-sheet harvesting23 and polydopamine (PDA) chemistry, a novel approach for constructing ECM coatings on metallic bone implants was proposed. Pre-osteoblasts, MC3T3-E1 cell-sheets were utilized to acquire ECM owing to its tissue-specific property.30 PDA, which is reported to be biocompatible24 and able to immobilize various biomolecules on materials’ surface25-26 without adverse effects on their biological properties,27-28 was used to immobilize ECM as surface coatings. The adhesion strength, morphology and components of ECM coatings were characterized and evaluated. Bone marrow mesenchymal stromal cell (BMSC), which is one of the most important sources of MSCs and has multi-lineages differentiation capability,29 was chosen as the cell model to investigate the regulatory effects on osteogenic differentiation of ECM coatings. 2. EXPERIMENTAL SECTION 2.1. Acquisition of MC3T3-E1 Cell Sheets. MC3T3-E1 cell sheets could be easily obtained through light-induced cell detachment method based on TiO2 nanodots films.23 Briefly, cells were cultured on TiO2 films for 7 days to be confluent cell sheets, and then illuminated with 365 nm ultraviolet for 20 min to detach them. In order to regulate the physical and biological cues of ECM in the cell sheets, after careful rinsing with phosphate buffer solution (PBS), the harvested cell sheets were treated with 4% paraformaldehyde for 10 min or 75% ethanol for 3 min. The treated cell sheets were further washed with PBS and deionized water for 5 times to remove the
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ACS Applied Materials & Interfaces
residual reagents. Finally, the resulted cell sheets were frozen at -20 oC and lyophilized for 12 h. 2.2. Fabrication and Characterization of ECM Coatings. As a commonly used bone implant material, Ti were employed as the substrate. Firstly, Ti substrates (99.9% Ti, 7 mm × 7 mm, 0.5 mm in thickness), were ultrasonically cleaned and acid-etched as described previously.31 Then, dopamine hydrochloride (Sigma) was added to Tris-HCl buffer solution (10.0 mM, pH 8.5) to obtain a dopamine solution (2.0 g/L). After that, Ti substrates were immersed in the dopamine solution with rigorous stirring at room temperature for 12 h. In order to expose the ECM components, the lyophilized cell sheets were turned over and placed on the substrates. Finally, the samples were allowed at 37 oC for 12 h to ensure the effective adhesion of cell sheets. Scanning electron microscope (SEM, Hitachi SU-70, working voltage at 3 kV) was used to observe the morphology. The chemical composition of different coatings was characterized with attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR, Thermofisher, Nicolet 5700) and X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD, Al Kα, 1486.6 eV). Surface topography features of different coatings were detected by atomic force microscope (AFM, NTEGRA Spectra, NTMDT) with a semi-contact-mode. A contact angle meter (Dataphysics, OCA20) was employed to determine the water contact angles (WCA) with a sessile drop method. The adhesion strength of the coating was measured on the universal testing machine using adhesive tapes (Minnesota Mining and Manufacturing, 3M610).32 ECM components in the coatings were detected through immunofluorescent staining. The samples were first washed with PBS and blocked using PBS with 2.5% BSA (Sigma) and 10% fetal bovine serum (Gemini) for 1 h at room temperature. Then, 0.5% BSA-PBS diluted rabbit anti-mouse primary antibodies (collagen I (Col-I), laminin (LN), Abcam) solution was added and the samples were incubated at 4 oC overnight. Afterwards, samples were washed and incubated with the secondary antibodies (Alexa Fluor 488, goat anti-rabbit) in PBS solution for 80 min at room temperature, and then washed with PBS and incubated with DAPI (Sigma), and finally imaged with confocal laser scanning microscopy (Zeiss LSM 780, Germany). 2.3. Cell Culture. Three-week old male Sprague-Dawley rats were used to isolate BMSCs.33 Briefly, after euthanasia, BMSCs were flushed out from the bone marrow of femurs and tibias. Then, they were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibico) containing 10% fetal bovine serum (Gibico), 0.272 g/L L-glutamine (Sigma) and 1% antibiotic solution (penicillin and streptomycin (Gibico)). The third to fifth passage cells were used in the follow-up experiments. The animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Zhejiang University, Hangzhou, China. 2.4. Cell Morphology. The BMSCs (20000 cells/cm2) were cultured on different coatings for 1, 4 and 7 days, and then rinsed and fixed in 4% paraformaldehyde. After that, the cells were dehydrated by a graded ethanol method and then dried in hexamethyldisilazane solution, and lastly observed with SEM. 2.5. Cell Viability and LDH Assay. Cell counting kit-8 (CCK-8) assay34 was used to determine cell viability after culturing for 1, 4 and 7 days with the seeding density of 20000 cells/cm2. At designed time point, the culture media were collected to detect the lactate dehydrogenase (LDH)
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release according to manufacturer’s procedure (Dojindo). Then, cells on samples were washed, and 10% CCK-8 solution was added and incubated at 37 oC for 3 h. Finally, the absorbance was read at 450 nm. 2.6. ALP Activity. The BMSCs (with a density of 20000 cells/cm2 for 7 days or 10000 cells/cm2 for 14 days) were seeded on different surfaces. After 4 days of culture, osteogenic induction medium was added for further cultivation. After incubation for 7 or 14 days, samples were washed and transferred to a new culture plate. Then, the cells were treated with cell lysis buffer (Sigma). The ALP activity was evaluated by measuring the absorbance at 405 nm and normalized to total protein contents, which were tested by a BCA protein assay. A BCIP/NBT ALP color development kit (Beyotime) was used to stain the ALP product of BMSCs on various samples. 2.7. Osteocalcin Secretion. The BMSCs were cultured on different coatings for 7 and 14 days. At designed time point, quantitative osteocalcin (OCN) activity was evaluated by Rat Osteocalcin Elisa Kit (Shanghai Westang Bio-tech.co.). The absorbance at 450 nm was measured and then normalized to the total protein contents. 2.8. Quantitative Real-time PCR Assay. The expression of osteogenesis-related genes was evaluated through real-time (RT) polymerase chain reaction (PCR) assay. The BMSCs with the density of 20000 cells/cm2 and 10000 cells/cm2 for 7 days and 14 days, respectively, were seeded on different coatings. The culture medium was changed to osteogenic induction media after 4 days of cultivation. At designed time point, the total RNA was extracted by TRIzol reagent. Then, RT-PCR was conducted on Roche Light Cycler 480 system with a SYBR Green I matermix. GAPDH, as the reference gene, was used to standardize the relative expression of target genes. The primers were displayed in Table S1. 2.9. Extracellular Matrix Mineralization. Mineral deposition on different coatings was evaluated by alizarin red staining. The BMSCs (10000 cells/cm2) were seeded on the samples. After 4 days of culture, the medium was replaced by osteogenic induction medium. At designed time point, the samples were washed and fixed in 4% paraformaldehyde, and then stained with 2% alizarin red. Afterward, the staining was desorbed with 10% cetylpyridinium chloride and the absorbance at 560 nm was read. 2.10. Statistical Analysis. All measurements were conducted in triplicate and the data were expressed as mean ± standard deviation (sd). Statistical analysis was determined via tukey’s post hoc test and one-way ANOVA. Student’s t-test was performed by SPSS software, and p value
