Structure and Property Changes in Self-Assembled Lubricin Layers

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Structure and property changes in self-assembled lubricin layers induced by calcium ion interactions George W. Greene, Rajiv Thapa, Stephen Andrew Holt, Xiaoen WANG, Christopher J. Garvey, and Rico F. Tabor Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03992 • Publication Date (Web): 18 Feb 2017 Downloaded from http://pubs.acs.org on February 19, 2017

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Structure and property changes in self-assembled lubricin layers induced by calcium ion interactions George W. Greene1‡,*, Rajiv Thapa2,‡, Stephen A. Holt3,‡, Xiaoen Wang1, Christopher J. Garvey3, Rico F. Tabor2 1

Institute for Frontier Materials and ARC Centre of Excellence for Electromaterials Science,

Deakin University, Geelong, VIC Australia, 2

3

School of Chemistry, Monash University, Clayton, 3800 Australia Australian Centre for Neutron Scattering, Australia Nuclear Science and Technology

Organization, Locked Bag 2001,Kirrawee DC, New South Wales 2232, Australia KEYWORDS: lubricin; PRG4; polymer brush; neutron reflectometry; self-assembly; adhesion; AFM; calcium; ion interactions

1 ACS Paragon Plus Environment

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Abstract

Lubricin is a “mucin-like” glycoprotein found in synovial fluids and coating the cartilage surfaces of articular joints which is now generally accepted as one of the body’s primary boundary lubricants and anti-adhesive agents. Lubricin’s superior lubrication and anti-adhesion are believed to derive from its unique interfacial properties by which lubricin molecules adhere to surfaces (and biomolecules, including hyaluronic acid and collagen) through discrete interactions localized to its two terminal end-domains. These regionally specific interactions lead to self-assembly behaviour and the formation of a well-ordered ‘telechelic’ polymer brush structure on most substrates. Despite its importance to biological lubrication, detailed knowledge about lubricin’s self-assembled brush structure is insufficient and derived mostly from indirect and circumstantial evidence. Neutron reflectometry was used to directly probe selfassembled lubricin layers confirming the polymer brush architecture and resolving the degree of hydration and level of surface coverage. While attempting to improve the lubricin contrast in the neutron reflectometry measurements, the lubricin layers were exposed to a 20 mM solution of CaCl2 which resulted in a significant change in the polymer brush structural parameters consisting of a partial denaturation of the surface binding end-domain regions, partial dehydration of the internal mucin domain ‘loop,’ and collapse of the outer mucin domain surface region. A series of AFM measurements investigating the lubricin layer surface morphology, mechanical properties, and adhesion forces in PBS and CaCl2 solutions reveals that the structural changes induced by calcium ion interactions also significantly alters key properties which may have implications to lubricin’s efficacy as a boundary lubricant and wear protector in the presence of elevated calcium ion concentrations.


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Introduction Lubricin (LUB) is a special type of mucin-like glycoprotein whose important role as a major biological boundary lubricant in articular joints is now well known and characterized.1-10 LUB’s excellent lubrication properties are derived primarily from its unique chemistry and molecular structure which gives rise to amphiadhesive properties and self-assembly behaviour.9-12 The LUB molecule possesses a unique tri-block structure consisting of a ~200 nm long, flexible, and highly hydrated central ‘mucin domain’ that is flanked on either end by relatively smaller but extremely adhesive globular ‘end-domains’.4

The mucin

domain of the molecule is heavily modified with approximately 2/3 of the amino acid residues within the mucin domain post-translationally modified with short, O-linked glycans which gives this domain a very strongly bound hydration layer that is primarily responsible for LUB’s excellent lubrication and anti-adhesive properties.4,

13

Compared with other

mucins, the glycosylation of the LUB mucin domain is relatively simple with more than 90% of the glycosylation consisting of the two glycans, -GalN-Gal and –GalN-GalN-Neu5Ac, in an approximate relative abundance of 33% and 66%, respectively.13 The terminal sialic acid residue (i.e. –Neu5Ac) on the more abundant –GalN-GalN-Neu5Ac glycan carries an anionic charge and, as a result, the mucin domain supports an overabundance of negative charge density. The end domains of the protein are not significantly glycosylated and contain sub domains similar to two proteins, somatomedin-B and homeopexin, known to play a special role in cell-cell and cell-extracellular matrix interactions, e.g., binding.4 These end domains are therefore extremely ‘sticky’ and are able to adhere to nearly all surface types (e.g. hydrophobic, hydrophilic, anionic, cationic, etc.)12 collagen14 and hyaluronic acid.1,

2, 8, 11, 15, 16

as well as to molecules such as

The adhesive end-domains and non-adhesive

mucin domain gives rise to both the aggregation of LUB in solution, with LUB dimers and trimers forming though adhesive coupling of end-domains9, as well as self-assembly


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behaviour in which LUB molecules are believed to adhere specifically to surfaces through both end-domains.10, 11

In addition to being an impressive biological lubricant, LUB exhibits a number of interesting properties including excellent anti-adhesive properties6,

12

and electrokinetic properties.17

These properties and the recent development of recombinant methods that allows LUB to be produced in large quantities has led to the development of LUB technologies for the treatment of arthritis and related conditions,18-20 eye lubrication,21 anti-adhesive coatings,12, 22, 23

and low-fouling microfluidic systems.17

Despite this interest and rapid increase in

technologies that take advantage of LUB’s interfacial properties, detailed knowledge of the self-assembled LUB layer structure is surprisingly sparse and has yet to be directly probed or visualized with most information inferred from indirect observations and circumstantial evidence.

The self-assembled LUB layer is believed to form a very specific, nano-structured, and highly ordered ‘telechelic’ polymer brush; essentially a polymer brush composed of molecular ‘loops’ where the molecule is grafted to the surface at both terminal ends rather than by a single end as is the case with more conventional brushes.9 Evidence for the telechelic brush structure of LUB originated from a series of Surface Forces Apparatus (SFA) experiments,9, 10 where it was observed that lubricin layers adsorbed to mica surfaces from solution consistently led to nearly identical repulsive force-distance curves with no apparent dependence on the concentration of lubricin in the solution. In these same experiments, it was observed that the onset of the repulsive force (on approach) was found to occur at a separation almost exactly ½ the molecular contour length of the protein (i.e. the end-to-end distance of the native protein rather than the full length of the peptide chain) when a LUB


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coated mica surface was compressed against an uncoated mica surface. Finally, it was found that the repulsive force curves obtained from the adsorbed LUB layers could be well-fitted using the well-known Alexandar-DeGenne polymer brush model.10

Although the synovial fluids of joints possess a similar electrolyte concentration as blood serum, one distinct difference is that the calcium ion concentration in synovial fluid is elevated by approximately 1.2 times that normally found in blood. In healthy synovial fluid, the concentration of Ca2+ ions have been reported to be up to 4.0 mM or more.24 Although experimental measurements or reports of the Ca2+ ion concentration in diseased joints (e.g. osteoarthritis, rheumatoid arthritis, gout) are difficult to come by, symptoms common to many joint diseases include the demineralization of the calcified zone of cartilage25, 26 and the appearance of precipitated crystals of calcium pyrophosphate27,

28

(and related compounds)

which strongly suggests these conditions are associated with higher than normal concentrations of ionic calcium. Ca2+ ions have been shown to bind to carboxylate moieties and have induced aggregation behaviour in glycoproteins such as fibronectin.29,

30

In

addition to glycoproteins, Ca2+ ions have also been implicated in the aggregation of relatively non-glycosylated proteins; most notably casein31 and secreted muscle foot proteins.32 Despite the importance of LUB to joint protection and joint health and the very large number of sialic acid residues within its glycome (the anionic charge on sialic acid is carried by a carboxylate group), there have been few studies investigating the potential interactions between LUB and Ca2+ and its effects upon the structure and properties of the molecule.

In this manuscript, we report the results of neutron reflectometry (NR) experiments performed on self-assembled layers of LUB on a silicon wafer which provides the first direct evidence confirming the telechelic polymer brush structure formed through adhesive


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attachment of the end-domain regions to a substrate. In addition, we exposed the selfassembled LUB layer to solutions of calcium chloride and observed a structural rearrangement of the adsorbed LUB layer mediated by calcium ion bridging interactions mostly through anionic charges carried by sialic acid residues located within the large, mucin domain regions of the protein. The NR model for the structural impact of Ca2+ ions was validated through a series of AFM experiments investigating the effects of Ca2+ ion on the LUB layer’s surface morphology, normal forces, mechanical properties, and adhesive properties.

2.0 Methods 2.1 Lubricin Purification LUB protein was purified using the procedure described in Greene et al12 (a slightly modified method based upon the protocols outlined in ref 4) from ~500 ml of bovine synovial fluid sourced from MC Herd (Corio; Victoria, Australia).

The synovial fluid was collected

percutaneously from the radiocarpal joints of freshly slaughtered cattle (~1 year old; male and female) using a sterile 18 gauge hypodermic needle and stored in a polypropylene bottle, on ice, until the time of processing (approximately 2 hours after collection). . The extracted and purified LUB was analysed for purity using a density gradient SDS-PAGE Biorad gel subsequently stained with coomasie blue. The relative purity of the LUB (as a fraction of the total protein content) was assessed using a Biorad imager and spectroscopic analysis and was found to be approximately 89%. The LUB band appeared on the SDSPAGE gel at approximately the 280 kDa region, consistent with previous reports.4 The concentration of LUB in the extracted solution was determined using the Biorad protein


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assay, with BSA as the standard. After the concentration of LUB was assayed, the solution was concentrated using a Millipore Amicon Ultra Centrifugal Filter with a 100 NMWL membrane to yield a final concentration of 400 µg/ml of protein in a buffer consisting of 25 mM Sodium Phosphate, 150 mM NaCl, 0.5 mM CaCl2 and 0.2 mM alpha lactose at pH 7.4. For experiments, this stock solution was diluted with additional PBS solution to the working concentrations as stated in the text.

2.2 Reflectometry Experiments Neutron reflectivity (NR) measurements were performed using the Platypus time-of-flight neutron reflectometer33 at the Australian Nuclear Science and Technology Organization (ANSTO; Lucas Heights, NSW Australia) which utilizes the cold guide CG3 at the OPAL 20MW research reactor. The NR measurements were performed with a polychromatic neutron beam (3 – 18 Å wavelength range) incident on the sample at angles (θ) of 0.8 and 3.8o. The slits defining the neutron beam were set such that the footprint of the beam on the surface was constant at 35 mm wide by 65 mm long for both incident angles with the instrumental resolution set at 3 %. A specular neutron reflectivity experiment measures the intensity of the neutrons reflected from the interface as a function of the momentum change perpendicular (Qz) to the surface with =

sin

and the reflectivity, R, is defined as

follows, = where II and IR are the incident and reflected beam intensities.

The raw NR data is then reduced to attain the reflectivity on an absolute scale. The neutron beam transmits through 100 mm of silicon which will have a varying transmission across the neutron wavelengths employed. For this reason direct beam measurements were acquired with the beam transmitted through the silicon at the slit settings appropriate for each angle. Data reduction then simply entailed dividing the reflected beam spectrum by the incident and 7 ACS Paragon Plus Environment

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then stitching together the two angular datasets based upon the overlap region between then. The absolutely scaled data could then be quantitatively analysed in terms of a layer model appropriate to the system. The modelling process applied assumes that the interface can be described as a series of slabs. At each of the interfaces in the system the neutrons are either refracted or reflected, with the reflected neutrons reaching detector and the refracted neutrons transmitted either to the next interface in the system or into the bulk solution. Each layer in the system is described by three parameters in the modelling process; that is, thickness, scattering length density (SLD), and interfacial roughness. The SLD depends on the density of the material and the nuclear scattering length of the materials nuclei. The model uses a Gaussian roughness to describe the change in SLD between layers.

The fitting was

performed using the Motofit macros within the Igor Pro environment.34 which uses the standard optical transfer matrix approach of classical optics35 to exactly calculate the reflectivity from a multilayer stack. A more detailed description of the slab model and fitting process employed can be found in refs 36 and 37. A genetic algorithm was employed to reduce the Chi squared value while iterating from the initial starting point to the final fit. In all cases the simplest model that described the data in a physically meaningful manner was selected.

The NR experiments were performed using a 100 mm diameter, 10 mm thick silicon block (Sil’tronix; Archamps, France) having an RMS roughness > 0.5 nm. Before coating, the block was cleaned using the RCA-1 method which involves submerging the block in a bath of 3:1:1 solution of DI water, 27% aqueous ammonia, and 30% hydrogen peroxide that was heated to 70º C for twenty minutes.

After the RCA-1 cleaning, the block was rinsed

thoroughly first with DI water and then with filtered isopropyl alcohol before undergoing a second cleaning cycle in a UV-ozone system for 20 minutes. The silicon block was then assembled into a flow cell utilizing a second silicon block (cleaned the same way as the first)


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separated by a flat, 100 micron thick polyester o-ring creating a dead volume in the cell of ca 630 μl. Through holes drilled onto either end of the second silicon block enables fluid to be flowed into the cell from one end and escape the cell at the other. For these experiments, fluid was injected either by hand using a syringe (for low volumes of lubricin solutions) or via a syringe pump (for higher volume buffers and solutions). Forming the LUB brush on top of the silicon was achieved by simply injecting a small volume of lubricin solution into the flow cell and allowing the LUB to naturally self-assemble into an ordered brush layer over a period of 15 minutes. After this self-assembly period, the unabsorbed LUB was rinsed out of the flow cell by flushing it with clean D2O PBS or CaCl2 solution, as appropriate. The details of the brush formation and the specific LUB solution volumes, concentrations, and order of injection are detailed in the section ‘3.1 Neutron reflectometry experiments’.

2.3 AFM experiments Triangular silicon nitride cantilevers (cantilever type D, MSCT, Bruker) with calculated spring constant of 0.042–0.046 N/m were used for force measurements (nominal resonant frequency, spring constant and length are 15kHz, 0.03 N/m and 225 µm respectively). Tips were not chemically modified. All AFM measurements were made using a JPK Nanowizard III AFM. Imaging and force spectroscopy curves were obtained in contact mode in PBS buffer initially, and then exchanged with a 20 mM CaCl2 solution, allowing for an hour of equilibration after solution exchange. 50 ul of 400 µg/ml lubricin were directly deposited on a glass surface (cover slips). After a period of 15 minutes, the glass surface was rinsed with copious amounts of clean and filtered PBS solution and placed in a petri dish filled also with PBS. For CaCl2 experiments, the PBS was exchanged with a 20 mM CaCl2 solution using 4 rounds of addition/extraction in the petri dish ensuring the sample was immersed at all times.


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The Hutter–Bechhoefer method38 was used to determine the actual spring constants of the cantilevers used. Experiments were carried out at room temperature and maximal set-point force of 1 nN was used for imaging. In order to ensure that representative force measurements corresponding to the lubricin coverage on the surface were obtained, force mapping was performed at different locations on the surface. Typical tip velocities of 1 µm/s or less were used, to avoid any influence of hydrodynamic drag on the very flexible cantilevers used. The mechanical response of the LUB layer was analysed using the Hertz/Sneddon model39, 40 for interaction of a sphere with a compliant material, assuming a spherical tip with radius of curvature 10 nm. The regions ascribed to the more compliant brush and more rigid end domains were fit separately. AFM data were analysed using the JPK Data Processing Version 6.0.26 from JPK instruments.

3.0 Results and Discussion

3.1 Neutron reflectometry measurements of LUB layers in PBS In order to obtain direct information about LUB’s conformational structure, neutron reflectometry (NR) measurements were performed on self-assembled LUB brushes adsorbed onto Si wafers from D2O PBS solution. The LUB brush was assembled in sequential stages inside a flow cell. Initially, the clean Si wafer was exposed to 1.5 ml of a 10 µg/ml solution of LUB which was injected into the flow cell by hand using a syringe over a period of approximately 1 min. At the end of the injection, there was an 8 minute ‘soak’ period in which the system was left idle to allow the LUB to adsorb to the surface. Following the soak period, the system was flushed with 4 ml of clean, filtered D2O PBS using a syringe pump and a flow rate of 0.5 ml/min to rinse away any unabsorbed LUB before the neutron reflectometry data was collected. Such a low concentration of LUB was used in this initial


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stage because it was hoped that structural information about a partially assembled (i.e. incomplete) LUB brush structure could be obtained and the self-assembly process could be observed at different stages. Unfortunately, due to weak scattering and poor contrast by the LUB layer in a D2O background, we were unable to resolve any LUB at the surface in this initial measurement. This injection and rinsing process was repeated a second and third time using 2 ml and 2.5 ml of 50 µg/ml solutions of LUB in D2O PBS respectively. By the third injection, neutron scattering by the LUB layer became apparent in the reflectometry plots (see Fig.1a). However, a fourth injection of 1 ml of a 120 µg/ml solution of LUB and a fifth injection of 0.35 ml of a 400 µg/ml solution of LUB (both in D2O PBS) did not result in any observable change in the neutron reflectometry curves indicating that, by the third injection, the self-assembly of the LUB brush was complete.

The reflectometry data collected for the third injection and shown in Fig. 1a was analysed by fitting the curve to a model in which the interface is described as a series of layers as described in section 2.2. As seen in the lack of strong fringes in the reflectometry curve, the adsorbed LUB layer did not give rise to a strong contrast. The reason for the poor contrast was determined to arise from the very heavy glycosylation of the mucin domain region which accounts for the majority of the protein’s length and gives this region a scattering length nearly identical to the D2O background. Given that the mucin domain layer is not seen at all, it is possible to estimate the overall D2O content of the layer at no less than 95% by volume (i.e. the content below which we would expect to get some scattering contribution from this layer); a hydration level in line with that reported for mucin gel structures41. The majority of the scattering obtained from the LUB layer in PBS therefore originates from the lightly glycosylated end-domain regions. From the model fitting, a plot of the scatter length density SLD as a function of distance from the Si interface was obtained which is shown in Fig.1b. 11 ACS Paragon Plus Environment

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From this model fit, a clear but very thin ‘interfacial’ layer determined to be approximately 6 nm thick was identified indicating a layer of surface adhered end-domains. Previous report estimate the size (i.e. diameter) of these globular domains at between 2-3 nm in diameter which was based upon the known hydrodynamic radii of the somatomedian B and hemopexin proteins which share much of their amino acid sequence with the end-domains.10

The

thickness of 6 nm which the NR measurements place for this layer strongly suggests that the diameter of the end-domains in LUB are, in fact larger, however it cannot be ruled out that a portion of non-glycosylated mucin domain very close to the end-domains may contribute some to this thickness. From the SLD of the interfacial layer it is possible to obtain the % surface coverage of the end-domains on the Si surface which was found to be approximately 15% or one molecule for every 190 nm2 with an average distance between end-domain anchoring points of 8.8 nm§.

This ‘grafting density’ agrees reasonably well with that

obtained in previous SFA experiments in which fit the repulsive forces to the AlexanderDeGenne polymer brush model42, 43 which yielded a surface coverage of ~ 5 mg/m2 and an average distance between adsorbed LUB molecules of approximately 10 nm.10

3.2 Neutron reflectometry measurements of LUB layers in CaCl2 In an attempt to achieve more contrast with the LUB mucin domain, an attempt was made to chemically dehydrate/condensate the molecule by exposing it to solutions of CaCl2 in D2O.

§

The % surface coverage is effectively, the measured SLD of the interfacial end-domain layer as a percentage of the expected SLD (calculated from the amino acid sequence) of an interfacial layer consisting of 100% enddomains. The area per end-domain and the distance between end-domains is based on a calculation that assumes 15% surface coverage and an hexagonal packing of spherical end-domains having a radius, r = 3 nm or ½ the end-domain layer thickness measured in the NR experiments. In this packing geometry, a whole enddomain occupies the center of a hexagon and 1/3 of an end-domain occupies each corner (for a total of 3 enddomains) within the area of a hexagon with sides of length a. The equation used was

! "

= #√# "

"

= 0.15. Solving this equation for length a gives the value a = 14.8 nm and an 2

area for the hexagon of 569 nm . The area per end-domain was obtained by dividing the area of the hexagon by 3 (the number of end-domain areas within this hexagon). The distance between end-domains was obtained by subtracting two end-domain radii from the length of the hexagon side, a.


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The ability of Ca2+ ions (and other divalent ions) to condensate proteins through the formation of ion bridging interactions is a well-known phenomenon with Ca2+ exhibiting a particular affinity to binding carboxylates30 which make up the vast majority of anionic charge carriers (i.e. sialic acid) in the LUB molecule; especially within the mucin domain region.

Indeed, Ca2+ binding to terminal sialic acid residues has been implicated in

facilitating the assembly of glycoprotein complexes including fibroin filaments.29, 44

For these experiments, a fresh layer of LUB was again deposited onto a freshly cleaned silicon wafer by directly pipetting 700 µl of a 400 µg/ml solution of LUB onto the wafer’s surface. After a period of 45 minutes, the wafer was rinsed with excess D2O PBS before assembling the flow cell for reflectometry experiments.

An initial reflectometry

measurement was performed in D2O PBS which reproduced the reflectometry curve obtained in previous experiments (and shown in Fig. 1a). Next, the D2O PBS was exchanged first with a 5 mM CaCl2 solution in D2O (4 ml at a flow rate of 0.5 ml/min) which showed no significant change in the NR curve. A second solution exchange was then performed using a 20 mM CaCl2 in D2O (4 ml at a flow rate of 0.5 ml/min) which gave rise to a significant change and the appearance of strong fringes as shown in Fig. 2a.

The NR data collected in the 20 mM CaCl2 solution was again analysed using the same model fitting procedure as used previously for the D2O PBS experiments and described in section 2.2. The results of the model fitting gave rise to three distinct layers defined by different SLD profiles. Again, a clear interfacial layer is visible that extends approximately 14 nm (140 Å) from the Si surface; approximately twice as large as was previously observed in the experiments with D2O PBS (see Fig. 1b).

Since the obtained thickness of the

interfacial layer appears to have doubled, it is likely that the Ca2+ ions have induced some


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denaturation of the end-domain regions of the LUB molecule. Above this interfacial layer, a much thicker layer of roughly uniform SLD becomes visible which was not previously observed in the D2O PBS and extends from the end of the interfacial layer out to a distance of ~ 84 nm (840 Å). This thicker layer is comprised of the central (i.e. internal) structures of the ‘mucin’ domain region of the molecule and is approximately 80% D2O sub-phase. This is a significant reduction in the D2O content within the mucin domain layer compared to the measurement performed in D2O PBS (which was determined to have a minimum of D2O content of 95%) indicating a partial, calcium induced dehydration. Above this central mucin domain layer is a thinner outer mucin domain layer, characterized by a steeply decreasing SLD, which possesses a thickness between 5-10 nm (50-100 Å) and a very high roughness as indicated by the manner in which the amplitude of the fringes observed at low Q values is rapidly damped. We note that the total thickness of the LUB layer in the CaCl2 solution was determined to be approximately 105 nm (1050 Å) which is essentially the same thickness previously reported in SFA experiments performed on LUB coated mica surfaces in PBS.10 Taken together, this three-layer structure is indicative of a swelling of the interfacial enddomains, partial hydration of the internal brush structure, and a significant collapse within the terminal surface region of the brush resulting in a poorly defined hydrogenous region. The collapse of the brush surface structure and dehydration within the central mucin domain is most likely brought about by the binding of Ca2+ ions with the carboxylate on terminal sialic acid residues (Neu5Ac). Being a divalent ion that sheds its hydration layer relatively easily, Ca2+ ions are known to form ‘calcium ion bridging’ interactions which are a type of physical cross-link formed when Ca2+ ions form coincident ionic bonds with two anions located on adjacent anionically charged macromolecules or within different regions of a single macromolecule. The apparent Ca2+ ion concentration dependence of the observed structural change is likely the manifestation of ion-condensation, which lowers the anionic charge


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density and electrostatic repulsion between LUB chains. The lack of apparent impact upon the LUB brush structure observed in the 5 mM CaCl2 solution is thus consistent with a concentration dependent reduction in the inter-chain electrostatic repulsion which prevents chains from approaching close enough for an ion-bridge to form below a threshold concentration.

To confirm the reversibility of Ca2+ ion interactions and the resulting

structural rearrangements, the 20 mM CaCl2 solution in D2O was again exchanged with D2O PBS and the measured NR curve again returned to the previously measured curve shown in Fig. 1a.

3.3 AFM investigation of the LUB brush structure and interfacial properties In order to shed more light upon the structural information obtained from the neutron reflectometry study, a complementary AFM study was performed to verify details of the modelling results, obtain missing information not achievable from the reflectometry measurements (e.g. total LUB layer thickness, LUB layer uniformity) and clarify the apparent structural rearrangement produced by the formation of Ca2+ ion bridging interactions.

For

this AFM study, layers of LUB were directly deposited from a 400 µg/ml solution in PBS onto a microscope slide (borosilicate glass) which is chemically very similar to the surface of a silicon wafer which always possesses a thin, native oxide (i.e. silica) surface film.

3.3.1 LUB layer morphology in PBS and CaCl2 solution AFM imaging was used to compare and contrast the surface morphologies of the uncoated silica substrate (Fig. 3a), the LUB layer morphology in PBS (Fig. 3b), and the same layer after the exchange of PBS with a 20 mM solution of CaCl2 (Fig. 3c). The LUB layer was first imaged in PBS solution immediately following deposition (see Fig. 3b) and shows an apparent conformal coverage with a remarkably smooth surface texture that gives an average


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root mean square (RMS) roughness of approximately 1.28 ± 0.14 nm which was calculated from 9 roughness measurements from 3 independent images taken at different positions on the sample surface (see Fig. 3d). We note that this roughness is not appreciably larger than the innate RMS roughness of the silica surface which was found to be ~ 0.47 ± 0.10 nm. The same LUB layer shown in Fig. 3b was imaged again after exchanging the PBS solution with a 20 mM solution of CaCl2 (see Fig. 3c). As seen in Fig. 3c, the conformal coverage of the LUB layer is preserved (i.e. there is no apparent desorption of LUB from the surface) but the measured RMS roughness of the layer increased significantly to a value of 3.81 ± 0.48 nm (see Fig. 3d). In addition to an increase in the RMS roughness, which is an averaged roughness, it should be noted that there is a distinct and noticeable change in the surface texture of the LUB layer compared with that previously observed in the PBS solution. While the ‘peaks’ and ‘valleys’ observed in the LUB layer surface appear small, more uniform in size, and randomly distributed in the PBS, the LUB layer surface in CaCl2 solution appears populated by clusters of large peaks, estimated to range between 5-14 nm in height. These large peaks appear to be scattered among larger patches of comparatively smoother LUB surface. This disorganized surface texture as well as the large height of the observed peak clusters, is consistent with the surface region collapse model obtained from the fitting of the NR data for CaCl2 solution (see Fig. 2b). Although the data is not presented here, it was observed that when the CaCl2 solution was exchanged back to PBS solution, the layer reverted back again to a similar structure as seen in Fig. 3a indicating that the structural effects of the Ca2+ ion interactions were fully reversible which is, again, consistent with the reversibility of the calcium-induced structural changes observed in NR measurements.

3.3.2 Mechanical properties of LUB layers in PBS and CaCl2 solutions


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It is clear from the change in the LUB layer’s RMS roughness that the surface morphology of the LUB layer is altered by Ca2+ ions interacting with the LUB molecules. To further investigate how the interactions of Ca2+ ions affect other properties of the LUB layer, a series of normal force measurements were performed on LUB layers in solutions of PBS and 20 mM CaCl2 using a low spring constant, flexible AFM cantilever with a sharp tip (radius of curvature 10 nm). First, comparing the normal forces on approach (see Fig. 4a and a’), a long range steric repulsive force is observed in both the PBS and CaCl2 solution that is consistent with previous SFA9, 10 and AFM11 studies performed on LUB layers in PBS assembled on mica and graphite substrates. Given the high ionic strength of the buffer solutions used, it is expected that any electrical double-layer force resulting from the charge on the LUB molecules would be extremely short range, as the typical Debye length in these solutions is 30 force curves), a gradual build-up of LUB on the AFM tip appears to occur, rendering the cantilever somewhat antiadhesive, which leads to a significant reduction (but not total disappearance) in the observed adhesive forces.

This reduction in the adhesion is always associated with a

significant increase in the range of the observed repulsive force measured on approach consistent with a LUB coated tip interacting with a LUB coated surface. For this reason, the number of force measurements that can be performed with a single cantilever is limited.

By integrating the adhesive force curves, it is possible to obtain the overall adhesion energy, which in these force measurements encompasses not only the energy of adhesion between the


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LUB molecules and the surface, but the free energy associated with unfolding and stretching of the molecule. Because the adhesion energy is path independent, comparing the adhesion energy makes it possible to identify differences in the initial (i.e. adhered and folded) state of the LUB molecule which would otherwise be obscured by the path dependence of the force measurement. Histograms of the adhesion energy measured from 34 force measurements performed in PBS and 34 force measurements performed in 20 mM CaCl2 solution are shown in Figs. 7a and b respectively.

Comparing the distributions of the measured adhesion

energies in Figs. 7a and b reveals a subtle but significant shift in adhesion energy measured in CaCl2 compared to PBS, with the mode value increasing slightly to 150-200 attojoules in CaCl2 from a value of 100-150 attojoules in PBS. Unfortunately, it is not possible to unequivocally determine the root cause for this apparent increase in the adhesion energy resulting from the interactions with Ca2+ ions. It is possible that the Ca2+ ions affect the LUB-surface adhesion by altering the interfacial electrostatic forces; however, it is equally possible that calcium ion bridging interactions within the mucin domain and/or the enddomains contribute to the extra energy required to unravel and eventually detach the molecules from the surface. However, given that Ca2+ ions appear to lead to some degree of denaturing in the interfacial foot region (see Fig. 2b), it is reasonable to speculate that the increase in adhesion energy observed arises from enhanced LUB-surface interactions as opposed to an increase in the force required to unfold the interfacial end-domains.

4.0 Conclusions The self-assembled ‘telechelic’ polymer brush structure adopted by LUB, though generally accepted9, 10 and supported by ample indirect and circumstantial evidence, has not previously been unambiguously resolved. Neutron reflectometry measurements of the self-assembled LUB brush in PBS indicate a clearly defined interfacial layer anchoring the LUB layer to the


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silicon surface that is approximately 6 nm thick; reasonably close, albeit larger than, the previously estimated diameters of the SMP-like and HPX-like end-domains9. Although NR was unable to resolve the mucin-domain region in PBS solution due to poor contrast, the fact that no contrast was achieved indicates an extremely high level of hydration in excess of 95% which is similar to the level observed in mucin gel structures.41 This high level of hydration implies that the LUB molecules must not only have a strongly bound hydration layer but also be reasonably ‘well spaced’ on the surface; a notion that is corroborated by the analysis of the interfacial layer that indicates a surface coverage of only 15% of the available silicon surface area. However, despite having such a low surface coverage, AFM images of the LUB layer surface morphology on silica (chemically similar to the surface oxide layer of silicon) in PBS indicates an incredibly smooth morphology that is barely ‘rougher’ that the underlying substrate. For a layer that previous SFA experiments9, 10 measure as approximately 100 nm thick, such a smooth surface, with an RMS roughness of ~1.28 ± 0.14 nm, can only be obtained if the two grafted end-domains within a single LUB molecule adsorb on the silica surface very close together and the mucin domain loop is as fully extended as possible. The consistency of the LUB layer thickness thus implies that the end-domains are already associated in solution prior to adsorption so that, when fully extended, the mucin domain loop length is roughly equal to ½ the molecular contour length.

NR reflectometry experiments also reveal a previously unreported structural change in the LUB brush induced by interactions with Ca2+ ions. The structural changes, which occur at calcium ion concentrations significantly above the level associated with ‘healthy’ synovial fluid (i.e. 5 mM) result in a collapse of the outer surface regions, a partial dehydration of the inner mucin domain regions, and a partial denaturing of the interfacial, end-domain region of the brush structure. Corroborating AFM data indicates that these structural changes are


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associated with a concomitant ‘roughening’ of the LUB layer surface morphology, an increase in the average ‘stiffness’ of the mucin domain layer, a decrease in the average ‘stiffness’ of the interfacial end-domain layer, and a slight enhancement in the apparent adhesion energy of LUB to the silica substrate. Adhesion force measurements consistently measure two maxima in the adhesion force separated by a deep ‘well’ of low adhesion force and distance roughly equal to the length of the mucin domain loop. This trend in the observed adhesion force is further evidence of the loop-like conformation of the surface adsorbed LUB molecules. It is currently not known whether or not calcium ion levels may reach sufficient concentrations, either as the result of injury or disease, to induce similar changes to LUBs structure and properties in vivo; or, if they do occur, what implications it would have on LUB’s ability to provide effective lubrication, wear protection, and/or antiadhesion to joint surfaces. The results of these experiments, however, strongly supports further investigation into calcium ion effects on the lubrication mechanisms of joints and articular cartilage and measurement of ionic calcium concentrations in arthritic synovial fluids.

5. Acknowledgments

This work was funded by the Australian Research Council through a Discovery Early Career Research Award (Project #: DE130101458). Dr. Greene would like to thank the ARC for their support with this award. This work was also supported by a research grant from ANSTO (grant #5406). The authors would also like to thank Dr Noelene Quinsey and the Monash Protein Production Unit, Clayton, Monash University, Victoria for their assistance with the purification of the native bovine lubricin protein. Dr. Greene also thanks M.C. Herd


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(Corio, VIC Australia) and Prof. Richard Fry (University of Melbourne) for their assistance in sourcing synovial fluid.

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of Surface Treatment Using Hyaluronic Acid and Lubricin on the Gliding Resistance of Human Extrasynovial Tendons In Vitro. J. Hand Surg. 2009, 34 (7), 1276-1281.

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M. A.; Blanchet, T.; Gleghorn, J. P.; Bonassar, L. J.; Bendele, A. M.; Morris, E. A.; Glasson, S. S., Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin. Arthritis Rheum. 2009, 60 (3), 840-847.


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Figure 1. Neutron reflectivity data collected for self-assembled LUB layers adsorbed to a Si surface in D2O PBS. (a) Plot of the reflectivity, R, vs the scattering vector, Q, showing both the raw data and the fit of the data obtained using the model fitting procedure described in section 2.2. (b) Plot of the scatter length density (SLD) as a function of the distance from the Si interface, D, obtained from the model fitting procedure described in section 2.2.


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Figure 2. Neutron reflectivity data collected for self-assembled LUB layers adsorbed to a Si surface in a 20 mM CaCl2 solution in D2O. (a) Plot of the reflectivity, R, vs the scattering vector, Q, showing both the raw data and the fit of the data obtained using model fitting procedure described in section 2.2. (b) Plot of the scatter length density (SLD) as a function of the distance from the Si interface, D, obtained from the model fitting procedure described in section 2.2.


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Figure 3. Representative AFM images showing the surface topography of (a) the SiO2 coverslip substrate and self-assembled LUB layers in (b) PBS solution and (c) 20 mM CaCl2 solution. The image shown in (c) was collected from the same LUB layer as shown in (b) after exchanging the PBS solution with the CaCl2 solution and equilibrating for 1 hour. All images were collected using a triangular silicon nitride cantilever with calculated spring constant of 0.042N/m-0.046N/m and tip of radius 10nm. (d) Representative height traces of the surface topographies of the SiO2 and LUB layers in PBS and CaCl2. The average RMS values shown were calculated from 9 total traces from 3 independent images (3 traces from each image) taken at different positions on the sample surface. A y-offset has been applied to the data for clarity.


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Figure 4. (a) Representative normal repulsive forces measure during approach for selfassembled LUB layers on a SiO2 surface in PBS and 20 mM CaCl2 solutions. (a’) The same normal repulsive force data shown in (a) plotted on a semi-log plot. (b,c) The normal force on approach (and separation) for a self-assembled LUB layer in PBS together with a schematic diagram illustrating the experimental geometry of the measurement at different stages of the measurement. Stage ‘0’ indicates the geometry where the tip is well away and not interacting with the LUB layer. Stage ‘1’ indicates where the tip is interacting predominanatly with the mucin domain ‘loops.’ Stage ‘2’ indicates the where the tip has penetrated deep into the LUB layer and the repulsive force is dominated by the mechanical properties of the interfacial end-domain region. The dashed lines in (c) and marked by ‘1’ and ‘2’ (corresponding to stages ‘1’ and ‘2’) show the fitting regions used in the determination of the long-ranged and short-ranged modulus values shown in Fig. 5 a-d.


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Figure 5. Histograms obtained for the long range modulus measured for a self-assembled LUB layer on a SiO2 surface measured in (a) PBS and (b) 20 mM CaCl2 solution. The longrange modulus is defined as the region of the repulsive force curve (see Fig. 4c) where the mechanical properties are dominated by the larger mucin domain region of the brush structure. The estimated error in the long range modulus measurements were ± 0.13 kPa for the PBS system and ± 0.08 kPa for the CaCl2 system. (c and d) Histograms obtained for the short-range modulus measured for a self-assembled LUB layer on a SiO2 surface measured in (c) PBS and (d) 20 mM CaCl2 solution. The short-range modulus is defined as the region of the repulsive force curve (see Fig. 4c) where the mechanical properties are dominated by the smaller interfacial end-domain region of the brush structure. The estimated error in the long range modulus measurements were ± 0.91 kPa for the PBS system and ± 0.56 kPa for the CaCl2 system.


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Figure 6. (a, a’, b, and b’) Representative force curves showing the repulsive forces on approach and adhesive forces upon separation of self-assembled LUB layers on SiO2 surfaces measured in (a and a’) PBS and (b and b’) CaCl2 solution. The ‘two peak’ trend shown in (a, a’, b, and b’) was observed in 15 out of 34 measurements (44%) in PBS solution and 12 out of 34 measurements (35%) in CaCl2. (c) Schematic illustration showing the interpretation and molecular responses leading to the repulsive and adhesive forces shown in (a). The regions of the force curve marked by red numbers 1-5 shown in (a) correspond to the stages marked with the same red numbers shown in (c).


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Figure 7. Histograms of the adhesive energy obtained by integration the adhesive forces measured upon separation for self-assembled LUB layers on SiO2 surfaces in (a) PBS and (b) 20 mM CaCl2 solutions. The estimated error in the adhesion force measurements were ± 14 attojoules the PBS system and ± 12 attojoules for the CaCl2 system.


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AUTHOR INFORMATION Corresponding Author *to whom correspondence should be addressed. Email: [email protected] Telephone: (+61) 3 924 68278 Author Contributions ‡

These authors contributed equally to this work.

GWG wrote the manuscript with

contributions from all authors. GWG, SAH, CCG, and RFT conceived of and designed the study. GWG, SAH, CCG, XW, and RFT executed the NR experiments. SAH, CJG, and GWG analyzed and interpreted the NR experimental results.

RT performed the AFM

experiments. RT, RFT, and GWG analyzed and interpreted the AFM experimental results. All authors have given approval to the final version of the manuscript. Funding Sources This work was funded by the Australian Research Council through a Discovery Early Career Research Award (Project #: DE130101458) and by a research grant from Australian Nuclear Science and Technology Organisation (ANSTO grant #5406).


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Structure and Property Changes in Self-Assembled Lubricin Layers

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