ARTICLE pubs.acs.org/Biomac
Colloidal Force Spectroscopy and Cell Biological Investigations on Biomimetic Polyelectrolyte Multilayer Coatings Composed of Chondroitin Sulfate and Heparin Steffi Grohmann, Holger Rothe, Marion Frant, and Klaus Liefeith* Institute for Bioprocessing and Analytical Measurement Techniques (iba), Rosenhof, 37308 Heilbad Heiligenstadt, Germany
bS Supporting Information ABSTRACT: To promote osteoblast adhesion and proliferation on (bio)material surfaces, biomimetic coatings resembling the natural extracellular matrix (ECM) are desirable. The glycosamino glycans (GAGs) chondroitin sulfate (CS) and heparin (HEP) are promising candidates for a biomimetic coating since they are two of the most prevalent noncollagenous biomolecules constituting the ECM. Coatings containing CS and HEP were prepared employing the “layer by layer” technique yielding polyelectrolyte multilayers (PEMs). Physicochemical and mechanical characterization of the coatings were performed by means of streaming potential measurements and colloidal force spectroscopy. The capability of the coatings to support cell adhesion, spreading, proliferation, and maintenance of an osteoblastic phenotype was assessed with SaOS osteosarcoma cells. We demonstrate that PEMs constructed from CS as the polyanion display a low Young's modulus correlated with poorly supported cell adhesion and proliferation. When the CS was adsorbed onto a stiffer polypeptide PEM basis, the Young's modulus increased, and the cell response was significantly improved. For HEP coatings an intermediate Young's modulus and moderate cell adhesion and spreading were observed. No significant changes in stiffness or cell response were detected when HEP was adsorbed onto the polypeptide film.
1. INTRODUCTION Surface properties of biomaterials can efficiently be modified with polyelectrolyte multilayer coatings (PEMs) to generate specific (biological) functionalities. PEM films deposited by the “layer by layer” technique are a well-established and highly recognized strategy to modify material surfaces.1,2 By simply designing the PEM construction or the two/three-dimensional distribution of the PEM film a wide variety of functions like cell adhesion, bacterial repulsion, protein adsorption, and coagulation can be controlled.214 Furthermore, PEM films may additionally be refined by incorporating drugs, antibiotics, growth factors, and/or nanoparticles into the coating to improve their biological functions.3,1518 Among the natural polyelectrolytes (i.e., polypeptides, polysaccharides, nucleic acids) glycosamino glycans (GAGs) and sulfated glycosamino glycans (sGAGs) are of special interest for designing PEM coatings for applications in bone contact. Both GAG and sGAG constitute most of the noncollagenous biomolecules of the extracellular matrix serving several pivotal functions. Heparin (HEP) is a sulfated GAG known to both be specifically bound by growth factors (like bone morphogenetic proteins)19 and to display anticoagulative properties. Chondroitin sulfate (CS, one COO and approximately 1.4 SO3 groups per disaccharide unit) is structurally very similar to HEP (one COO and approximately three SO3 groups per disaccharide r 2011 American Chemical Society
unit). Furthermore CS is reported to aid the mineralization process on collagen fibrils.2022 The combined advantages of (i) to bind growth factors from the surrounding interstitial fluid and (ii) to facilitate in vivo mineralization make both CS and HEP interesting candidates for preparing bioactive multilayer coatings. However, to create biomimetic coatings containing both calcium phosphates and growth factors the cell biological compatibility of the PEM films composed of sGAG has to be verified first. Thus, the aim of this study was to characterize the cellular adhesion, spreading, and proliferation of osteoblast-like cells cultured on biomaterial coatings containing the extracellular matrix molecules CS and HEP. The mechanisms and principles of anchorage-dependent cells to sense the mechanical properties of their physiological environment and their cell biological and molecular biological response toward the varying substrates' stiffness (their surrounding) is a fascinating new research field addressing both cell biologists and material scientists.2325 A generally accepted hypothesis is that every cell type favors a substrate stiffness similar to the mechanical properties of the ECM surrounding the cell. Thus, cells of neuronal and musculoskeletal origin favorably Received: August 17, 2010 Revised: April 12, 2011 Published: April 14, 2011 1987
dx.doi.org/10.1021/bm200258q | Biomacromolecules 2011, 12, 1987–1997
Biomacromolecules attach, spread, and proliferate on soft (Pa range) and stiff (kPa to MPa range) substrates, respectively.26 The choice of the polyelectrolytes (PE) utilized for film construction strongly affects the properties of the generated coating. Thus, films composed of PE with a high water binding potential, that is, GAG, will result in highly swellable, hydrogel-like coatings displaying low stiffness.9,27,28 Since a low Young's modulus is strongly correlated with reduced cell adhesion and proliferation of cells of musculoskeletal origin, effective ways of stiffening these soft ECM analogous PEM films are sought to promote osteoblast adhesion and proliferation. One approach of preparing surfaces with a controlled elasticity from highly hydrated PEM films is the chemical cross-linking of carboxyl and amino groups by means of carbodiimide chemistries.9,29 Furthermore, attempts of photo cross-linking films by introducing photoreactive functionalities were reported.3032 However, all of these technologies are based on the chemical modification of the natural polyelectrolytes. In the case of photo cross-linking photoreactive groups like acrylates or benzylic acid have to be introduced into the polymer which may alter their function and the PEM build-up process. Finally, cross-linking the films with harsh chemicals is timeconsuming, and cytotoxic agents may not be rinsed from the film completely. This paper is the first report on the mechanical properties of PEM films composed of chondroitin sulfate and heparin serving as polyanions in film construction. Additionally, coatings are prepared that deliver biologically active GAG molecules on an underlying stiffer PEM basis composed of biodegradable polypeptides (no chemical pre- or post-treatments were conducted). Cell biological analyses with osteoblast-like cells were performed to evaluate the cytocompatibility of the resulting coatings.
2. MATERIALS AND METHODS 2.1. Multilayer Film Preparation. All chemicals were purchased from Sigma (Germany) in the highest purity available and used without further purification unless stated otherwise. Poly-Llysine (PLL, MW 3070 kDa), fluorescein isothiocyanate labeled PLL (PLL-FITC, MW 3070 kDa), poly-L-glutamic acid (PGA, MW 50100 kDa), chondroitin sulfate (CS, MW ∼63 kDa), and heparin (HEP, MW 1719 kDa) were dissolved in a Hepes/NaCl buffer (25 mM Hepes, 137 mM NaCl, pH 7.4) at a concentration of 1 mg/mL and sterile filtered (0.2 μm) before use. Films were constructed by alternating the deposition of the polycation (PLL) and polyanion (PGA, CS, or HEP) layers onto the substrate either manually or employing a dipping robot (DR3, Riegler and Kirstein, Berlin, Germany). The incubation time for each polyelectrolyte solution was 5 min followed by three cycles of rinsing with Hepes/NaCl buffer. Borosilicate glass discs (B33, diameter 15 mm, thickness 0.7 mm, Schott Jena AG, Germany) were coated for cell biological and topographical analysis. For mechanical characterizations by means of colloidal force microscopy coated silicon wafers were utilized. Initially the substrate surfaces were cleaned with detergents (SDS) and ultra pure water (Millipore). Thereafter, the surface was activated by etching with either 0.1 M HCl or 35% HNO3 (v/v). 2.2. Reflectometric Interference Spectroscopy (RIfS). RIfS analyses of the PEM film construction were performed with the BIAffinity system (Analytik Jena AG, Germany). The SiO2 surface of RIfS transducers (Analytik Jena AG, Germany) was preactivated by incubation with 100 μL of 35% HNO3 for 10 s followed by extensive rinsing with deionized water before they
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were mounted within the system. All buffers and solutions employed were filtered (0.2 μm) and degassed. At a buffer flow rate of 10 μL/min the activated transducer was rinsed inside the system until a stable baseline was obtained. PEM film deposition always initiated with injecting 30 μL of PLL (at 10 μL/min) followed by a rinsing time of at least 5 min. All further injections were always carried out with 30 μL of the respective PE at a flow rate of 10 μL/min until the final film architecture was received. Data are presented as the shift of a maximum (570670 nm) in the interference spectrum corresponding to the optical thickness of the resulting PEM film. 2.3. Zeta Potential Measurements. The surface charge was analyzed via streaming potential measurements in a SurPASS system equipped with an adjusting gap cell (Anton Paar GmbH, Graz, Austria) according to published studies.33,34 The confinement of the flow cell was achieved through two parallel and exchangeable B33 glass substrates (20 mm 10 mm 0.7 mm). To monitor the change in zeta potential during film assembly, the PE layers were deposited onto the substrates inside the gap cell. Briefly, the chamber was assembled by mounting the glass substrates within the flow cell. Preactivation of the surfaces was performed by injection of 0.1 M HCl and subsequent extensive rinsing of the cell with Hepes/NaCl buffer. Then 400 μL of PE were injected and left to adsorb for 5 min. Finally, the chamber was washed gently four times with 1 mL buffer, and the resulting surface charge was measured. This procedure was repeated until the desired film architecture was achieved. A 1 mM NaCl electrolyte at pH ∼ 5.5 was employed to determine the streaming potentials. Pressure profiles from 0 to 300 mbar were applied in two flow directions. Streaming potentials were measured and converted to the corresponding zeta potentials according to Fairbrother Mastin.35 To investigate the physicochemical properties of the PEM films employed for the cell biological investigations, zeta potentials over a pH range were recorded. PEM coated glass samples were mounted within the gap cell. The channel width was adjusted to 120 μm. The electrolyte (1 mM NaCl) was adjusted to pH 3 with 0.1 M HCl, and zeta potential measurements were performed after automated titration of the electrolyte with 0.1 M NaOH (20 μL increments) in a pH range of 3.09.5. Streaming potentials were converted to zeta potentials as described above. 2.4. Atomic Force Microscopy (AFM). Both topographical and colloidal force analyses of the PEM films on glass substrates were performed with an atomic force microscope (Nanowizard I, JPK, Germany) equipped with a micro fluidic cell (Smart Cell, JPK, Germany) rinsed with Hepes/NaCl buffer at 23 °C. For topographical investigations unmodified cantilevers cleaned in an argon plasma prior to use were employed (Si3N4, tip radius
