Nanoscale Conformation and Compressibility of Cartilage Aggrecan Using Microcontact Printing and Atomic Force Microscopy

Introduction. The nanomechanical behavior of cartilage extracellular matrix (ECM) macromolecules has received increasing attention1-4 since ECM degradation leads to tissue and joint dysfunction with age and arthritis.5 For example, loss of the branched proteoglycan, aggrecan, and its highly negatively charged constituent glycosaminoglycan (GAG) polymer chains (Figure 1) significantly reduces cartilage’s compressive stiffness.5 The nanoscale force between opposing endgrafted GAG layers (a system that mimics local molecular responses to tissue deformations) has been measured over a broad range of pH and ionic strength (IS) solution conditions2 and was well described by a Poisson-Boltzmann-based, electrostatic double layer (EDL) “charged rod” model.6 In vivo, however, GAGs are covalently linked to a core protein, creating a dense cylindrical “bottle-brush” architecture, thus motivating studies on intact aggrecan, which is a closer-to-physiological model system. Here, we probe the conformation and compressibility of an aggrecan-functionalized surface by combining the techniques of microcontact printing (μCP7,8) and contact mode atomic force microscopy (AFM) (Figure 1). μCP enabled the creation of a patterned surface with densely packed, chemically endgrafted aggrecan confined to well-defined micrometersized surface areas and a hydroxyl-terminated selfassembling monolayer (OH-SAM) filling the rest of the surface area. By AFM imaging over boundaries between aggrecan and OH-SAM regions, the height and, hence, the deformation of the aggrecan were directly measured as a function of aqueous bath conditions (IS ) 0.001-1 M, pH ) 3-7) and normal compressive load (0-40 nN). Experimental Section. Fetal bovine aggrecan9 (0.5 mg/mL) was solubilized in deionized (DI) water with 1 μM dithiobis(sulfosuccinimidyl propionate) (Pierce #21578), which can react with amine groups at the N-terminal and lysines at the Nand C-termini of the core protein, thus forming disulfide bonds. These disulfides were reduced to thiols by reacting with dithiothreitol (Pierce #150460) in 0.1 mM aqueous solution for 1 h. Excess reactants were removed by spinning at 3500 rpm overnight with a centrifugal filter (Centricon, Millipore, 10 kDa cutoff). OH-aggrecan patterned substrates were made by μCP7 of 4 mM 11-mercaptoundecanol, HS(CH2)11OH (Aldrich), in ethanol on a freshly cleaned 1 cm × 1 cm gold-coated substrate10 for 30 s using a hexagonal patterned poly(dimethylsiloxane) (PDMS) stamp (hexagon side length ) 15 μm and spacing ) 15 μm) followed by incubation in 50 μL of 1 mg/mL thiol-terminated aggrecan solution for 48 h. Hence, the aggrecan was located within the hexagons and the OH-SAM outside the hexagons. OH-functionalized AFM gold-coated probe tips (Digital Instruments, k ∼ 0.06 N/m, tip radius ∼50 nm) were prepared by immersion in 4 mM 11-mercaptoundecanol for 48 h to minimize electrostatic interactions between the probe tip and aggrecan layer. Aggrecan height in aqueous solution was measured as the relative height between the OH-SAM (negligible thickness ∼1-2 nm) and aggrecan (Figure 1) regions under different compressive normal forces using a Multimode Nanoscope IV AFM (Digital Instruments) in contact mode in fluid at a scanning frequency of 1 Hz. Solutions were 0.001-1 M NaCl in Milli-Q water (pH ∼5.6) and 0.01 M phosphate buffered saline (PBS) at pH ) 3, 7, and 10 and 0.154 M PBS at pH ) 7.4. After AFM experiments were completed, aggrecan grafting density on the surface was assessed by removal of the aggrecan via boiling the substrate in DI water for 60 min. The residual solution was lyophilized and redissolved in 50 μL of DI water. A dimethylmethylene blue (DMMB) dye binding assay11 yielded a total aggrecan surface density equivalent to ∼10.1 ( 1.4 mg/m2, or one aggrecan molecule per ∼20 nm × 20 nm. The GAGs from this fetal bovine aggrecan have contour lengths of ∼40 nm.9 Results and Discussion. Aggrecan height was found to be nonhysteretic (independent of previous solvent conditions and loading history) and decreased markedly with increasing IS from 0.001 to 1 M under constant normal compressive load (Figure 2) and with increasing normal load at constant IS (Figure 3a). The maximum height, ∼310 nm in 0.001 M NaCl and ∼0 nN normal force, was ∼78% of the known contour length of the aggrecan used (∼400 ( 60 nm).9 The strong dependence of aggrecan height on IS indicates that electrostatic interactions are a critical determinant of the nanomechanical behavior of aggrecan. The carboxyl and sulfate groups on the CS-GAG are ionized at all bath pH and IS of Figure 3.10 The electrostatic interaction distance in monovalent electrolyte solutions is determined by