Nanoassemblies of Tissue-Reactive, Polyoxazoline Graft-Copolymers

Nanoassemblies of Tissue-Reactive, Polyoxazoline Graft-Copolymers Restore the Lubrication Properties of Degraded Cartilage Giulia Morgese,†,‡ Emma Cavalli,‡ Mischa Müller,‡ Marcy Zenobi-Wong,*,‡ and Edmondo M. Benetti*,† †

Laboratory for Surface Science and Technology, Department of Materials, and ‡Cartilage Engineering + Regeneration Laboratory, Department of Health Sciences and Technology, ETH Zürich, Zürich, Switzerland S Supporting Information *

ABSTRACT: Osteoarthritis leads to an alteration in the composition of the synovial fluid, which is associated with an increase in friction and the progressive and irreversible destruction of the articular cartilage. In order to tackle this degenerative disease, there has been a growing interest in the medical field to establish effective, long-term treatments to restore cartilage lubrication after damage. Here we develop a series of graft-copolymers capable of assembling selectively on the degraded cartilage, resurfacing it, and restoring the lubricating properties of the native tissue. These comprise a polyglutamic acid backbone (PGA) coupled to brush-forming, poly-2-methyl-2-oxazoline (PMOXA) side chains, which provide biopassivity and lubricity to the surface, and to aldehyde-bearing tissue-reactive groups, for the anchoring on the degenerated cartilage via Schiff bases. Optimization of the graft-copolymer architecture (i.e., density and length of side chains and amount of tissue-reactive functions) allowed a uniform passivation of the degraded cartilage surface. Graftcopolymer-treated cartilage showed very low coefficients of friction within synovial fluid, reestablishing and in some cases improving the lubricating properties of the natural cartilage. Due to these distinctive properties and their high biocompatibility and stability under physiological conditions, cartilage-reactive graft-copolymers emerge as promising injectable formulations to slow down the progression of cartilage degradation, which characterizes the early stages of osteoarthritis. KEYWORDS: graft-copolymers, osteoarthritis, cartilage engineering, surface assembly, nanofilms

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contrast, at low shear velocities and high contact pressures, the fluid is squeezed out from the synovium and lubrication takes place by means of macromolecular complexes of polysaccharides and glycoproteins, including hyaluronic acid (HA), aggrecan, and lubricin,5−9 acting as natural boundary biolubricants at the cartilage surface.2,10 Natural or traumatic modifications of the lubrication mechanisms in articular joints are correlated with their overall

atural lubrication mechanisms occurring at the articular cartilage surface provide very low friction (μ < 0.01) at the joint articulation and ensure no wear.1 These extraordinary tribological properties have been increasingly investigated over the last two decades, and the most accepted theory on joint lubrication nowadays relies on the synergistic action of boundary and fluid lubricants.2,3 Depending on loading frequencies, their magnitude and sliding speeds, interstitial fluid pressurization or boundary lubrication can occur.4 The first mechanism dominates at high shear velocities and low contact pressures, when the fluid film in the joint is thicker than the asperities of the cartilage surface. In this regime, the synovial fluid mainly supports the total load. In © 2017 American Chemical Society

Received: November 22, 2016 Accepted: March 8, 2017 Published: March 8, 2017 2794

DOI: 10.1021/acsnano.6b07847 ACS Nano 2017, 11, 2794−2804

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Figure 1. PGA-α-PMOXA(x)-β-HBA synthesis and application. (a) Cationic ring-opening polymerization of 2-methyl-2-oxazoline was initiated by methyltriflate (i) and terminated by ammonia (ii), yielding PMOXA-NH2, which was coupled to the PGA backbone through NHS/DCC chemistry (iii). HBA was then coupled to the remaining free carboxylic acid groups of the backbone through PFP/DCC (iv). (b) PGA-α-PMOXA(x)-β-HBA featuring different side-chain density (α) and length (x) and density of tissue-reactive HBA functions (β) were immobilized on degraded cartilage through Schiff-base linkages between the amino groups exposed on the cartilage surface and the aldehyde functions at the HBA groups.

polyglutamic acid (PGA) backbone, which can readily react with the degraded cartilage surface via Schiff-base formation, and poly-2-methyl-2-oxazoline (PMOXA) side chains, which form a dense and highly hydrated “brush” layer, capable of reestablishing the lubrication properties of the native cartilage. This layer also protects surfaces from contamination of biomolecules from the synovial fluid. The design of the PGA-PMOXA graft-copolymer takes inspiration from natural lubricating macromolecules such as lubricin and mimics the structure of synthetic graft-copolymers, which were previously applied on model inorganic surfaces to form lubricious and biopassive polymer brushes.24−26 In addition, the choice of PMOXA as the main constituent of the proposed graft-copolymer formulation is highly relevant, since this hydrophilic and biocompatible polymer has been increasingly studied as a promising, chemically durable alternative to poly(ethylene glycol) (PEG) in biomaterials and drug-delivery applications.27−29 The immobilization of the graft-copolymers on the degraded cartilage surface via Schiff bases, which replace the hydrophobic and/or electrostatic interactions occurring between natural biolubricants and the articular cartilage,30 is shown to be essential for the development of a robust boundary layer. Simultaneously, the formation of a dense PMOXA−brush interface on the degraded cartilage enables a substantial reduction in the COF to the values typically observed for the native tissue. Remarkably, the proposed synthetic, cartilage-reactive graftcopolymer is demonstrated to act, itself, as a boundary lubricant for the articular cartilage, without requiring the recruitment of other biomacromolecules from the synovial fluid. These highly attractive features, combined with the in vivo stability and biocompatibility displayed by the prepared graft-copolymers, make them a promising formulation for treating the early stages of OA, when the progression of the disease could be slowed down through an efficient tissue protection and the restoration and maintenance of cartilage lubrication.

longevity, while it was demonstrated that an increase in the coefficient of friction (COF) of the cartilage causes an exponential decrease of the lifetime of the knee.11 An alteration of the articular cartilage composition or changes in the content of biolubricants in the synovium are associated with an increase in friction and wear, progressively leading to severe, degenerative diseases of the articular joint, such as osteoarthritis (OA).12 Remarkably, the degradation of the extracellular matrix (ECM) correlated to OA, combined with the absence of vascularization in the articular cartilage, cause an irreversible progression of the disease, which ultimately represents one of the leading sources of adult disability nowadays.13,14 With the aim of halting OA at its early stages, the most developed clinical strategies have been aiming at restoring the lubrication properties of the cartilage. This has been attempted by viscosupplementation using intra-articular injections of corticosteroids and HA, although the lack of relief durability and the need for subsequent injections have made this approach controversial.15,16 Alternatively, research efforts were made to modify the cartilage surface with reactive polymer species intended to recruit HA and lubricin from the synovial fluid17−19 and restore a lubricious molecular layer on the tissue. Nevertheless, the newly formed macromolecular films can still be degraded by enzymes present in the osteoarthritic joints, e.g., matrix metalloproteinases (MMPs) and hyaluronidases,20 and thus would be ineffective during OA. This represents a relevant drawback, since while the adsorption of biomacromolecules on the cartilage surface plays a crucial role in its lubrication properties, the damage and/or disruption of the lubricin and HA layers by the action of enzymes cause an increase in friction and the occurrence of wear.21−23 In order to circumvent these limitations and to propose an injectable formulation that could halt cartilage degradation during OA, we report here on the synthesis and application of a fully synthetic biolubricant, which resists enzymatic degradation and can bind on the surface of the osteoarthritic cartilage, restoring the lubrication properties of the native tissue. This is based on a graft-copolymer featuring an aldehyde-bearing, 2795


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α are correlated with a higher concentration of HBA groups (β), the increment of copolymer reactivity was shown to favor the formation of thicker films. In addition, longer PMOXA side chains presumably hindered the accessibility of the aldehyde functions of the HBA groups, causing the chemisorption of fewer copolymers for a given PMOXA density α. The formation of the copolymer films on aminolized silicon oxide was additionally tested by a quartz crystal microbalance with dissipation (QCM-D). By comparing the swollen thicknesses of the films obtained by QCM-D with the corresponding dry thickness values measured by VASE, the water content (expressed as volume %) was estimated for each copolymer type. As reported in Table 2, the amount of swelling water

RESULTS AND DISCUSSION Synthesis of the Brush-Forming Copolymers and Application on Model Surfaces. Cartilage-reactive, brushforming copolymers include a multifunctional backbone based on PGA, onto which PMOXA side chains are alternatively grafted together with hydroxybenzaldehyde (HBA) functions (Figure 1a). When contacting the degraded cartilage surface, the HBA units covalently bind to the amino groups exposed on the collagenous tissue through Schiff-base formation, whereas the side polymer segments form a lubricious and biopassive brush interface (Figure 1b). By varying the density of PMOXA segments at the copolymer backbone (α), their length (x), and the density of tissue reactive HBA groups (β), a library of nine different PGA-α-PMOXA(x)-β-HBA graft-copolymers (which will be generally termed as PGA-PMOXA-HBA) featuring different side-chain crowding and backbone reactivity were thus synthesized (Table 1).

Table 2. PGA-PMOXA-HBA Film Parameters PGA-αPMOXA(x)β-HBA

Table 1. Analytical Data of the Synthesized GraftCopolymers PGA-α-PMOXA(x)-β-HBAa

αb

xb

xc

βd

PGA-0.1-PMOXA(30)-0.9-HBA PGA-0.3-PMOXA(30)-0.7-HBA PGA-0.6-PMOXA(30)-0.4-HBA PGA-0.1-PMOXA(100)-0.9-HBA PGA-0.3-PMOXA(100)-0.7-HBA PGA-0.6-PMOXA(100)-0.4-HBA PGA-0.1-PMOXA(120)-0.9-HBA PGA-0.3-PMOXA(120)-0.7-HBA PGA-0.6-PMOXA(120)-0.4-HBA

0.13 0.31 0.64 0.11 0.34 0.62 0.14 0.32 0.61

27 27 27 95 95 95 121 121 121

26 26 26 91 91 91 116 116 116

0.71 0.43 0.21 0.62 0.31 0.14 0.45 0.24 0.11

α

x

0.1

30 100 120 30 100 120 30 100 120

0.3

0.6

dry thickness (nm)a 1.5 1.1 0.99 1.1 0.86 0.8 0.96 0.76 0.7

± ± ± ± ± ± ± ± ±

0.1 0.1 0.08 0.1 0.03 0.1 0.03 0.04 0.1

hydrated thickness (nm)b

water content (vol %)c

± ± ± ± ± ± ± ± ±

68 70 80 84 86 86 82 83 88

4.7 3.7 4.9 6.9 6.1 5.7 5.3 4.4 5.9

0.7 0.9 0.5 0.8 0.3 0.4 0.5 0.3 0.8

a

Measured by VASE (mean values and standard deviations were calculated preparing three replicates per sample and measuring five points on each of them). bMeasured by QCM-D (mean values and standard deviations were calculated preparing two replicates for each sample). cEstimated from the comparison between VASE and QCMD.

a α, x, and β expected from the feed ratios of PGA, PMOXA, and HBA during the synthesis. bCalculated from 1H NMR spectroscopy, as exemplarily shown in Figures S1−S3 in the Supporting Information. c Measured by GPC analysis (Table S1 in the Supporting Information). d Estimated by 1H NMR spectroscopy and UV−visible spectroscopy.

showed a slight increment following the increase of density and length of PMOXA segments (increase of α and x), indicating the formation of more hydrated adlayers with increasing the relative MOXA content in the graft-copolymers. Interestingly, the increases of PMOXA side-chain density and length were also accompanied by the formation of more uniform copolymer films, as demonstrated by atomic force microscopy (AFM) (see Figure S5 in the Supporting Information). More reactive and less hindered adsorbates (lower values of α and x) can react promptly to the amino-bearing surface via multiple Schiff-base binding. This causes the deposition of thicker but also more heterogeneous films (Figure S6 in the Supporting Information), due to the possible formation of surface-grafted “loops”32 and the limited capability of rearrangements provided by copolymer backbones strongly bound to the surface via a large number of covalent anchors. In contrast, bulkier and less reactive copolymers (higher values of α and x) formed thinner but more uniform films, as displayed by the AFM micrographs (Figures S5 and S6 in the Supporting Information). To evaluate substrate shielding toward protein contamination by PGA-PMOXA-HBA films, graft-copolymer-coated samples were exposed to full human serum (FHS), and the amount of adsorbed proteins was later measured as dry thickness increment by VASE (Figure 2a). An increase of PMOXA grafting density α and chain length x resulted in a

Cartilage degeneration during early OA is accompanied by the depletion of the components of its surface layer (lubricin, glycosaminoglycan (GAG), and HA) and the concomitant exposure of collagen amino groups at the tissue surface.31 In order to investigate the surface reactivity of PGA-PMOXAHBA species toward aminolized surfaces and the properties of the originated graft-copolymer films, we first tested the adsorption of the different copolymers on model substrates based on (3-aminopropyl)trimethoxysilane (APTES)-functionalized silicon oxide substrates. Following overnight incubation, X-ray photoelectron spectroscopy (XPS) confirmed the formation of covalently bound copolymer adlayers via Schiffbase linkages between the HBA groups at the copolymer backbones and the amino groups exposed on the functionalized silicon substrates (Figure S4 in the Supporting Information). The robust chemisorption of PGA-PMOXA-HBA graftcopolymer on aminolized surfaces was also confirmed by control experiments where PGA-PMOXA copolymers (not presenting aldehyde groups) could not be deposited on APTES-functionalized silicon substrates and where PGAPMOXA-HBA did not bind to native silicon oxide wafers (in the absence of amino groups at the substrate). Variable-angle spectroscopic ellipsometry (VASE) measurements on the different copolymer films showed a progressive decrease of the film thickness with increasing the PMOXA density α and the length of the side polymer chains (x). Since lower values of 2796


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Figure 2. Protein resistance of PGA-PMOXA-HBA films as a function of PMOXA grafting density α and chain length x. The biopassive properties of the different graft-copolymer films deposited on APTES-derivatized silicon oxide substrates were evaluated as dry thickness increments by VASE following exposure to FHS (a). FITC-albumin adsorption on graft-copolymer-functionalized, degraded cartilage tissues was measured by means of fluorescence microscopy and microplate reader (b). Protein adsorption was normalized for the FHS dry thickness and FITC-albumin fluorescence measured on APTES-functionalized silicon oxide and degenerated cartilage tissue, respectively. All the experiments were performed in triplicates, and mean values and standard deviations were calculated measuring five points for each sample.

Figure 3. Synthesis and application of FL_PGA-PMOXA-HBA on degenerated cartilage tissue. (a) FL_PGA-PMOXA-HBA graft-copolymers were synthesized by functionalizing the free carboxylic acids of PGA with a 95:5 molar ratio of HBA to FL (i and ii). The native bovine cartilage was digested by ChABC (iii), and FL_PGA-PMOXA-HBA was finally chemisorbed on the degraded tissue surface (iv). (b) Microplate reader and (c) fluorescence microscopy confirmed the formation of FL_PGA-PMOXA-HBA films on the degraded cartilage surface (FC) when compared with untreated digested cartilage (DC).

responsible for the progressive cartilage destruction.33 In the early stages of OA, these processes are limited to the degradation of GAGs and aggrecan molecules, which leads to the exposure of the collagen layer,34 making the ε-amino groups of the lysine content of the collagen accessible on the surface (Figure 3). In order to mimic closely this specific condition, native cartilage samples extracted from bovine knees were digested by chondroitinase ABC (ChABC) before incubation in the copolymer solution.35−37 This degradation process caused the depletion of the lubricin layer and cleavage of the GAGs, yielding a tissue surface with very similar composition to the early osteoarthritic cartilage (Figure S11 in the Supporting Information).38−41 Degraded cartilage samples were subsequently used as substrates for testing the chemisorption of fluorescently labeled PGA-PMOXA-HBA graft-copolymers by fluorescence microscopy. Specifically, PGA-PMOXA-HBA species were modified by introducing fluorescein (FL)

progressive depletion of protein adsorption, with copolymer films presenting the highest α of 0.6 and the longest PMOXA side chains (x = 120), providing a nearly full biopassivity. These results indicate that an increase of hydration and surface coverage by more crowded and less surface-reactive copolymers are determining factors in conveying antifouling properties to the graft-copolymer coatings. In contrast, more reactive and less hindered copolymers formed thicker films presenting defects. These films displayed a lower amount of swelling water due to both the low concentration of PMOXA segments and the large number of Schiff-base linkages that anchor them onto the substrate. All these features translated into a less efficient protection of the surface from protein contamination. Application of PGA-PMOXA-HBA Copolymers on Degraded Bovine Cartilage Surfaces. During OA progression, the content of proteoglycans and collagen within the articular cartilage is reduced31 and matrix-degrading enzymes produced by osteoarthritic chondrocytes and synoviocytes are 2797


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detected only to a depth of 8 ± 2 μm after 1 day of graftcopolymer incubation. After 7 days, the penetration depth increased only to 20 ± 6 μm, indicating that the FL_PGAPMOXA-HBA remains on the surface of the tissue. Moreover, the chondrocytes’ viability at day 7 was 88% (normalized to the control), which is comparable with the value recorded after seeding the cells on top of the PGA-PMOXA-HBA films. In order to investigate the in vivo stability of the chemisorbed graft-copolymers, similar scaffolds functionalized with FL_PGA-PMOXA-HBA were implanted subcutaneously in nude mice for 21 days, using untreated scaffolds as negative controls (see Methods and Supporting Information for details). During this implantation time, no macroscopic signs of inflammation or adverse effects were recorded and, following sacrifice of the animals, the size of the explanted scaffolds remained unmodified (Figure S8f in the Supporting Information). The high in vivo stability of the copolymer coatings was demonstrated by measuring the fluorescence emission of the explanted scaffolds, which displayed comparable values to the fluorescence of a duplicate simultaneously incubated within a physiological medium at 37 °C. Furthermore, no fluorescence emission was detected from the surrounding tissues, indicating that no desorption of the graft-copolymers from the collagen supports took place (Figure S10 in the Supporting Information). Microscopically, the histological analysis on the explanted scaffolds and the surrounding tissues did not show differences between the control and the copolymer-coated samples in terms of both inflammation and fibrous capsule size (Figure S8b−e in the Supporting Information). It is noteworthy to mention that compared to the previously proposed surface modifiers for cartilage,17,18 PGA-PMOXAHBA graft-copolymers cannot be degraded by enzymatic reactions typically taking place within osteoarthritic joints20 and involving the cleavage of specific amino-acid sequences48,49 or glycosidic bonds50,51 that are absent in PGA-PMOXA-HBA. This distinctive feature makes them a very promising injectable and fully synthetic lubricant for the treatment of diseases of the articular cartilage. Lubrication Properties of PGA-PMOXA-HBA Films on Degraded Bovine Cartilage Surfaces. Investigating the tribological properties of the degraded cartilage following the application of PGA-PMOXA-HBA copolymers is particularly relevant, since therapies aimed at restoration of the natural lubrication of cartilage are currently lacking for the treatment of OA. To this end, degraded cartilage samples were treated with PGA-PMOXA-HBA and placed against each other in a custombuilt tribometer that allowed the recording of the COF at different sliding speeds and under different applied loads (Figure 4). Since knee joints support contact stresses varying from 0.5 to 5 MPa52 during their typical activities, we reproduced pressures ranging from 0.5 to 0.7 MPa by applying normal loads of 5 and 10 N (see Methods and the Supporting Information for details) at a sliding speed of 5 mm·s−1. In order to better simulate the natural joint environment, the tribological experiments were performed within a bovine synovial fluid solution of the same graft-copolymers applied to the degraded cartilage surface. The obtained COF values were subsequently compared to those recorded sliding two native cartilage (NC) samples against each other and analogous degraded cartilage (DC) surfaces, both immersed in pure bovine synovial fluid. Immunodetection by Western blotting confirmed the presence of lubricin in the synovial fluid (Figure

functions along the PGA backbones (to yield FL_PGAPMOXA-HBA, as depicted in Figure 3a), this dye featuring a high fluorescence quantum yield (0.92 in water42) and a maximum emission wavelength (512 nm in water) that does not interfere with the cartilage autofluorescence.43,44 The very low loading of FL on the PGA assured no remarkable changes in both the reactivity and the adsorption kinetics of the graftcopolymers, as experimentally proved on model aminolized surfaces. Following incubation in FL_PGA-PMOXA-HBA, the treated cartilage showed high intensity of fluorescence emission compared to an uncoated control, confirming the efficient functionalization of the tissue surface (Figure 3b and c). Remarkably, the graft-copolymer attachment is specific for the degraded tissue, whereas no copolymer film was formed on the native cartilage surface when it was incubated in the same copolymer solutions, as demonstrated by the fluorescence micrographs reported in Figure S7 in the Supporting Information. Fluorescence microscopy could not be applied to evaluate the copolymer film thickness and the tissue coverage for the different copolymer types. Nevertheless, passivation of the digested cartilage and its dependency on the graft-copolymer parameters (i.e., α, β, and x) could be indirectly evaluated by measuring the adsorption of proteins on the graft-copolymermodified tissues. This was tested by monitoring the adsorption of fluorescently labeled albumin (a globular protein that is abundant within the synovial medium),45 following similar experimental procedures to the ones used for the protein adsorption tests on graft-copolymer-functionalized model surfaces. As shown in Figure 2b, digested cartilage functionalization generally reduced the amount of absorbed proteins. Although a very similar trend of protein adsorption among the different graft-copolymer films was observed, if compared to similar coatings applied on aminolized model surfaces, protein contamination was typically more pronounced. This was probably due to the intrinsic surface roughness of the digested cartilage tissue (see histology data reported in Figure S11),46 which is believed to hinder the formation of homogeneous copolymer adlayers. On these substrates, the primary adsorption of proteins represented the main source of surface contamination, taking place at the coating defects that expose the underlying tissue surface.47 Nonetheless, it is remarkable that films originating from the highest copolymer grafting density showed the most pronounced reduction of albumin physisorption (less than 20% compared to an untreated, degraded tissue surface). Hence, also in the case of digested cartilage, densely grafted and less reactive graft-copolymers could protect the underlying tissue from an extensive protein physisorption. The biocompatibility of PGA-PMOXA-HBA films was additionally tested both in vitro and in vivo. We used copolymer-coated ChondroGide collagen scaffolds to evaluate bovine chondrocyte viability by live/dead assay (see Methods for details). As shown in Figure S8a in the Supporting Information, cell viability above 87% was recorded on the modified scaffolds, indicating a high biocompatibility for all the graft-copolymer types. In addition, we further determined the penetration depth of the graft-copolymers into live explanted bovine cartilage plugs and the influence of graft-copolymer diffusion through the tissue on the viability of chondrocytes. As reported in Figure S9 in the Supporting Information, FL_PGA-PMOXA-HBA was 2798


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The possible detachment of these weakly bound layers during the tribotests was evaluated by applying fluorescently labeled FL_ PGA-0.6-PMOXA(120)-0.4-HBA on the degraded cartilage and subsequently measuring the fluorescence emission of the synovial solution. As no emission could be detected, we excluded this hypothesis. Nevertheless, we interpreted these results as a consequence of the reduced shielding capability exerted by thinner coatings from more crowded graftcopolymers when these are subjected to relatively high pressures. In addition, this phenomenon could be coupled to a reversible detachment of the crowded copolymers and a subsequent surface rearrangement into a less uniform film. A substantial improvement in lubrication with low COF values was recorded for all the graft-copolymer films presenting PMOXA densities lower than 0.6, i.e., for copolymers more strongly bound to the cartilage surface. Further analysis of the friction data revealed how the structural parameters of the graftcopolymers determined the tribological properties of the treated cartilage. In particular, it was possible to establish a correlation among number of PMOXA repeating units ([MOXA]) of the graft-copolymers constituting each film, the recorded COF, and the corresponding protein repellence shown by the specific coating (Figure 5). Protein repellency and lubrication by brush-forming adsorbates represent two properties that have been often correlated, both being generated by the entropic shield by a dense brush assembly, coupled with the enthalpic barrier produced by its hydration capability.54−56 Due to the distinctive morphological and chemical properties of the cartilage surface, which make it far from being an ideally flat and uniform substrate, the correlation between protein adsorption and lubrication for this particular system becomes even more significant and biologically relevant. In addition, as previously mentioned, a fine control over the adsorption of biomolecules on the cartilage would represent a further tool to hamper the progression of OA, as the action of enzymes from the synovium could be suppressed through the application of a protein repellent layer. As reported in Figure 5, increasing the surface concentration of MOXA units leads to a progressive decrease of bioadhesion, the films with [MOXA] ≥ 7000 showing more than 80% reduction of protein adsorption compared to the uncoated cartilage. In contrast, the measured COF comparatively increased at both the applied loads tested, generally following an opposite trend. This result could be explained considering that a MOXA-rich surface efficiently repelled the adsorption of proteins from solution, although this was not a sufficient condition to guarantee a low COF on the articular cartilage. The effective reduction of friction on such a substrate required

Figure 4. Tribological properties of graft-copolymer-functionalized degraded cartilage. Degraded cartilage slices were coated with PGA-PMOXA-HBA graft-copolymers, and the COF values were measured sliding them against each other using a bovine synovial fluid solution of the corresponding graft-copolymer at 5 mm/s (a− b−c) and applying two different normal loads (5 and 10 N). On each graph, the recorded values of COF are reported for the different copolymer grafting densities (α) and a fixed side-chain length: x = 30 for (a), x = 100 for (b), x = 120 for (c). Native cartilage (NC) and digested cartilage (DC) were chosen as positive and negative controls, respectively.

S11 in the Supporting Information); thus the tribological properties of graft-copolymer-treated cartilage could be correlated with systems realistically mimicking the articular cartilage with its synovium components. As shown in Figure 4a−c, all the graft-copolymer coatings reduced the COF of the DC under the tested conditions with values ranging between 0.007 and 0.1. It is remarkable that the copolymer-treated cartilage also showed better lubricating properties than the NC,53 with the exception of PGA-0.6PMOXA(120)-0.4-HBA under a load of 5 N and all the PGA0.6-PMOXA-0.4-HBA at a 10 N load. For all the graftcopolymer films investigated, the COF was stable for 60 min of measurements, indicating no detectable wear of the films, while the recorded values were highly reproducible among several cartilage samples modified with the same graft-copolymer. Interestingly, an increase in the density of PMOXA chains on the copolymer backbone (for α = 0.6), i.e., a decrease of the capability by the copolymers of binding to the tissue surface, translated into higher COF values and thus worse lubricating properties by the films.

Figure 5. Protein repellency and tribological properties of graft-copolymer films on degraded cartilage as a function of the number of PMOXA repeating units [MOXA] under applied loads of 5 N (a) and 10 N (b). The dashed black line corresponds to the COF value of native cartilage under the different sliding conditions, and the pink circles highlight the [MOXA] of the best performing graft-copolymers in terms of both antifouling and lubricating properties. 2799


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was terminated by adding 23 mL (11.7 mmol) of a 0.5 M solution of NH3 in THF at room temperature, and the polymerization mixture was left under stirring for other 48 h. The solvent was removed under reduced pressure, and the crude solid product was dissolved in the minimum amount of Milli-Q water and purified by dialysis against water using 500 Da cutoff tubes. PMOXA(30)-NH2 (7.1 g, 79% yield) was obtained after freeze-drying as a white powder. Using the same procedure but varying the 2-methyl-2-oxazoline to methyl triflate ratio (100:1 and 120:1) and using 1 kDa cutoff dialysis membranes for the purification, PMOXA(100)-NH2 and PMOXA(120)-NH2 were obtained in 71% and 74% yield. The coupling between the PGA backbone, PMOXA(x)-NH2, and 4hydroxybenzaldehyde was carried out in two steps and, in the first step, varying the molar concentration of PMOXA(x)-NH2 and PGA to yield three different copolymer grafting densities (α = 0.1, 0.3, 0.6, with α defined as the number of PMOXA side chains per glutamate unit). As an example, we report the synthesis of PGA-α-PMOXA(30) with α = 0.3. PGA-H (290 mg, 2.23 mmol of carboxylic acid groups) was dissolved under an inert atmosphere in 10 mL of dry dimethylformamide (DMF) and dicyclohexylcarbodiimide (DCC) (460 mg, 2.23 mmol). N-Hydroxysuccinimide (NHS) (256 mg, 2.22 mmol) and 4dimethylaminopyridine (DMAP) (270 mg, 2.23 mmol) were added after complete dissolution in 5 mL of dry DMF, each. After 24 h of stirring at room temperature always under an inert atmosphere, 1.7 g (0.8 mmol) of PMOXA(30)-NH2 dissolved in 10 mL of dry DMF was added, and the solution was left under stirring for 48 h, prior to precipitation in DEE, dissolution of the solid in Milli-Q water, and dialysis against water using 12−14 kDa cutoff tubes. PGA-0.3PMOXA(30) was obtained as a white, fluffy solid after freeze-drying (1.05 g, 66% yield). Later on, HBA was coupled to PGA-0.3PMOXA(30) at the remaining free carboxylic acid functions of PGA via pentafluorophenol (PFP)-DCC-mediated coupling, finally yielding PGA-α-PMOXA(x)-β-HBA. In particular, 300 mg (1.8 um) of PGA0.3-PMOXA(30) was dissolved in 10 mL of dry DMF under an inert atmosphere; 65 mg (0.35 mmol) of PFP and 73 mg (0.35 mmol) of DCC were subsequently added (after dissolution in 5 mL of dry DMF each) under stirring at 0 °C. The solution was left stirring at room temperature for 20 h, and 48 mg (0.39 mmol) of DMAP and 48 mg (0.39 mmol) of HBA (after dissolution in 5 mL of dry DMF each) were added. After 48 h of stirring at room temperature under an inert atmosphere, the solvent was evaporated and the solid was dissolved in Milli-Q water. After filtration to remove the insoluble side products of the reaction, the solution was purified by dialysis against water using 12−14 kDa cutoff tubes. PGA-0.3-PMOXA(30)-HBA was obtained as a white, fluffy solid after freeze-drying (200 mg, 61% yield). 1 H NMR spectroscopy was employed to verify the copolymer molecular architecture and to determine the PMOXA chain length x and grafting density α (see Supporting Information for the details about the method). Fluorescently labeled PGA-α-PMOXA(x)-β-HBA (FL_PGAPMOXA-HBA) copolymers were synthesized using a slightly modified procedure, namely, by complementing HBA with 5 mol % of fluorescein in its free acid form. Gel Permeation Chromatography (GPC). GPC measurements were performed at 40 °C with an Agilent high-pressure liquid chromatography setup, equipped with a 1260 refractive index detector and a column set of two PLgel 5 μm mixed D columns and similar guard column (Agilent) in series. The eluent used was N,Ndimethylacetamide containing 50 mM LiCl, at a flow rate of 0.6 mL/min. Poly(methyl methacrylate) (PMMA) standards were used to determine the molar mass values. 1 H NMR Spectroscopy. NMR spectra were recorded on a Bruker Avance III 500 or 700 MHz at room temperature, using D2O as solvent for PMOXA(x)-NH2 and dimethyl sulfoxide-d6 (DMSO-d6) for PGA-α-PMOXA(x)-β-HBA. 1H NMR was used to confirm the composition of the different graft-copolymers and calculate the PMOXA chain lengths and grafting densities (details are explained in the Supporting Information).

the formation of thicker and more robustly anchored coatings, which were generated by graft-copolymers presenting a lower concentration of side PMOXA chains and a concomitant higher content of tissue-reactive HBA groups. The best compromise, in terms of both lubrication and biopassivity, was observed for 5000 ≤ [MOXA] ≤ 7000 and especially for films generated from PGA-0.3-PMOXA(120)-0.7HBA and PGA-0.3-PMOXA(100)-0.7-HBA copolymers, for COF measured at 5 and 10 N, respectively. These graftcopolymers, which presented “average” PMOXA grafting densities, were capable of reducing protein adsorption up to 80% and lower the COF by 80% with respect to the DC and by 50% compared to the NC. Even though, within a natural joint as well as in the studied system, cartilage lubrication is mainly due to synergistic effects between fluid and boundary mechanisms,2,57,58 what is clearly emerging from this study is the capability of PGA-PMOXAHBA copolymers to passivate the degraded tissue surface, efficiently restoring the natural lubrication of articular cartilage and acting exclusively as boundary lubricants. Remarkably, control tribological tests demonstrated that PMOXA-based graft-copolymers without the tissue-reactive groups do not act as fluid lubricants, and the presence of anchors for the cartilage surface is essential for them to perform as friction reducers (see note in ref 59 for further details).59 Hence, tissue-reactive PGAPMOXA-HBA copolymers represent a fully synthetic replacement for the natural boundary lubricants such as lubricin, effectively rehabilitating cartilage lubrication after degradation without the need to recruit biomacromolecules from the synovial fluid.

CONCLUSIONS PGA-PMOXA-HBA copolymers react with the degraded cartilage through Schiff-base linkages, efficiently resurfacing the tissue. A systematic tuning of the copolymer architecture demonstrated that more sterically hindered and less reactive species form a PMOXA-brush barrier that strongly reduces protein contamination on the treated cartilage but is not able to restore lubrication on the degraded cartilage. In contrast, PGAPMOXA-HBA films with intermediate PMOXA side-chain density and higher reactivity toward the tissue surface not only reduce protein adsorption but also restore the lubrication properties of cartilage after degradation, often showing lower COF values compared to the native tissue. All these attractive features, coupled with their high biocompatibility and in vivo stability, make PGA-PMOXA-HBA graft-copolymers promising candidates for the development of treatments to halt or slow down the progression of OA. METHODS PGA-α-PMOXA(x)-HBA Synthesis. Monodisperse 30 kDa poly(Lglutamic acid) sodium salt was purchased from Alamanda Polymers Inc. (Huntsville, AL, USA) and converted into the corresponding free acid (PGA-H) using a proton exchange resin (Amberlite IR-120). Amino-terminated PMOXA (PMOXA(x)-NH2) was synthesized by cationic ring-opening polymerization (CROP) of 2-methyl-2-oxazoline (MOXA) (Sigma-Aldrich) as previously reported,60 using methyltriflate (Sigma-Aldrich) as initiator and a 0.5 M solution of NH3 in THF (Acros Organics) as terminating agent. Namely, 2-methyl-2-oxazoline (10 g, 118 mmol, 30 equiv, distilled from KOH) was dissolved in dry acetonitrile (25 mL) under a N2 atmosphere. Methyl triflate (645 mg, 3.9 mmol, 1 equiv) was added at 0 °C under N2. The mixture was heated to 70 °C and kept at this temperature for 24 h under stirring and within an inert atmosphere. After this time, the polymerization 2800


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Two crystals for each graft-copolymer film were used to calculate the mean values and standard deviations. X-ray Photoelectron Spectroscopy. XPS was performed using a VG Theta Probe (Thermo Fisher Scientific, East Grinstead, UK) spectrometer. The spectra were acquired using a monochromatic Al Kα source with a beam size of 400 μm (power of 100 W) in the constant-analyzer-energy mode. During the acquisition, the flood gun was kept on to compensate the charges. A pass energy of 100 eV and a step size of 0.1 eV were applied. The raw spectra were processed using CasaXPS software (version 2.3.15, Casa Software Ltd., Wilmslow, Cheshire, UK). The background subtraction was performed using the Shirley method. After background subtraction, the peaks were fitted using the product of Gaussian and Lorentzian functions. Atomic Force Microscopy. The morphology of PGA-α-PMOXA(x)-β-HBA copolymer films was studied with a Bruker Dimension Icon atomic force microscope in tapping-mode, employing a SiN cantilever with a resonance frequency of 312 kHz and a normal spring constant of 24 N/m. Protein Adsorption Studies. In order to test the resistance toward protein contamination by PGA-α-PMOXA(x)-β-HBA films, copolymer adlayers deposited on model APTES-silicon oxide surfaces were exposed to full human serum (Precinorm U, Roche Diagnostics GmbH, Mannheim, Germany) solutions. The copolymer adlayers were initially hydrated by immersion in HEPES II for 5 min, and then they were subsequently exposed to undiluted human serum for 15 min. During incubation, the samples were kept under ambient conditions without stirring. Following exposure to serum, the samples were rinsed with HEPES II and ultrapure water, dried under a stream of N2, and analyzed by ellipsometry. APTES-functionalized silicon oxide surfaces were used as controls. A similar experimental procedure was applied for studying protein adsorption on PGA-α-PMOXA(x)HBA-functionalized cartilage samples. The physisorption of fluorescein isothiocyanate conjugate (FITC)-labeled albumin (SigmaAldrich) was evaluated by means of fluorescence microscopy (Axioskop 2 Plus Carl Zeiss microscope equipped with an Alexa 488 filter) and hybrid microplate reader (Synergy H1, BioTek, Winooski, VT, USA). In Vitro Cytotoxicity Studies. ChondroGide scaffolds of 2 mm in diameter (Geistlich Pharma AG) were prepared by using a sterile biopsy punch. The scaffolds were immersed overnight in 0.1 mg/mL graft-copolymer solution in PBS, washed twice with PBS, and freezedried. The coated scaffolds were subsequently transferred inside agarose-coated 24-well plates and seeded with bovine chondrocytes at passage 3 (250 000 cells/scaffold) in a total volume of 5 μL of cell suspension per punch. Cell-loaded scaffolds were left at room temperature (RT) for 10 min before adding 1 mL of medium (Dulbecco’s modified Eagle’s medium, Glutamax, 10% fetal bovine serum, 50 μg/mL L-ascorbic acid, and 1% penicillin−streptomycin) in each well. The entire plate was then incubated for 24h at 37 °C. Chondrocyte viability was tested using the LIVE/DEAD protocol. Briefly, the scaffolds were incubated with 6.6 μg/mL propidium iodide, 2 μM calcein AM, and 10 μg/mL Hoechst 33342 at 37 °C for 1 h. Afterward, the scaffolds were washed twice with PBS, incubated for 1 h in fresh medium, and finally washed once with medium. All the experiments were done in triplicates, and scaffolds exposed to PBS only were used as control. Three images for the scaffold were acquired with a Zeiss ApoTome.2 fluorescence microscope, and image analysis was performed using Fiji. In order to evaluate the cytotoxicity of the graft-copolymers not bound to the cartilage surface, live explanted bovine cartilage plugs (2 mm in diameter) were incubated for 7 days in medium supplemented with the graft-copolymers at a concentration of 0.1 mg/mL. The chondrocytes’ viability was assessed via a Trypan Blue (Invitrogen, T10282) exclusion assay after digestion of the cartilage plugs with collagenase (1.2 mg/mL), using a Countess automated cell counter. In Vivo Experiments. Scaffolds of 6 mm diameter were obtained from sterile ChondroGide (Geistlich Pharma AG) using sterile biopsy punchers. The punches were immersed overnight in 0.1 mg/mL FL_PGA-0.6-PMOXA(100)-HBA solution in PBS and washed twice with PBS. Punches treated only with PBS were used as controls. In vivo

UV/Visible Spectroscopy. In order to determine the content of HBA functions within the graft-copolymers, UV absorption spectra of 0.1 mg/mL graft-copolymer solutions in DMSO were recorded at room temperature on a Jasco V660 Japan UV/vis spectrophotometer in the range of 200 to 800 nm. The HBA quantification (expressed as β) was considered as the number of aldehyde functions per glutamate unit. Aminolized Substrates. In order to study the adsorption of PGAα-PMOXA(x)-β-HBA on aminolized model surfaces, silicon oxide substrates exposing primary amino groups were used. Specifically, silicon wafers (Si-Mat, Germany) were cleaned for 15 min in a piranha solution (3:1 mixture of concentrated H2SO4 and H2O2; warning: piranha solution is very reactive and corrosive; use extreme caution!), extensively washed with ultrapure water and absolute ethanol, and finally blow-dried under a stream of nitrogen gas. The samples were subsequently silanized by vacuum deposition inside a chamber containing few drops of APTES. Following APTES layer formation, the substrates were rinsed with absolute ethanol, annealed in an oven at 55 °C for 1 h, and freshly used for the graft-copolymer adsorption. Bovine Cartilage Extraction and Digestion. Articular cartilage was extracted from bovine knees of 1-year-old calves (Slaughterhouse Zürich) and incubated at 37 °C in the presence of 1 U/mL ChABC in 0.01% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) solution for 1 h. This enzymatic treatment was shown to allow the exposure of the collagen layer.61 Control samples were soaked for the same period in PBS. After incubation, the samples were washed three times with a BSA solution and once with ultrapure water. Copolymer adsorption experiments were performed applying PGA-α-PMOXA(x)β-HBA solutions on freshly digested cartilage substrates. Preparation of PGA-α-PMOXA(x)-β-HBA Films. Aminolized silicon wafers and digested cartilage samples were immersed overnight in 0.1 mg/mL HEPES II (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid +150 mM NaCl, adjusted to pH 7.4) solutions of PGA-α-PMOXA(x)-β-HBA, washed extensively with ultrapure water, and finally dried under a stream of N2. Variable Angle Spectroscopic Ellipsometry. Thickness measurements were performed using an M-2000F variable-angle spectroscopic ellipsometer from J. A. Woollam Co. (Lincoln, NE, USA). All data were recorded at a wavelength range between 370 and 1000 nm using focusing lenses at 70° from the surface normal. The raw ellipsometric data were analyzed with WVASE32 software using a three-layer model (Si/SiO2/Cauchy; An = 1.45 and Bn = 0.01, Cn = 0). All measurements were performed under ambient conditions. Three substrates for each graft-copolymer film were prepared, and five points for each sample were measured to calculate the mean values and standard deviations. Quartz Crystal Microbalance with Dissipation. The hydrated thickness of the graft-copolymer films was measured by QCM-D using an E4 instrument (Q-Sense AB, Göteborg, Sweden) equipped with dedicated Q-Sense AB software. SiO2-coated crystals (LOT-Oriel AG) with a fundamental resonance frequency of 5 MHz were used as substrates. Before the experiment, the substrates were cleaned by 15 min of sonication in toluene and 2-propanol, 30 min of UV-ozone cleaning (UV Clean model 135500 from Boekel Industries, Inc.), and again 15 min of onication in toluene and 2-propanol. After cleaning, the crystals were dried under a stream of N2 and functionalized with APTES as previously described. All the solvents used were degassed for 20 min. The modified crystals were exposed to HEPES II at 25 °C until a stable baseline was established. Later on, the buffer was replaced with a graft-copolymer solution until complete adsorption was obtained. To determine the stability of the film and eliminate the physisorbed polymers, washing steps with HEPES II were performed. Regeneration of the crystals was accomplished through an overnight immersion in 2% (v/v) sodium dodecyl sulfate (SDS) solution. The values of hydrated thickness for the different graft-copolymers were obtained by applying a Voigt extended viscoelastic model62 to fit the frequency and dissipation shift, using three overtones. By comparing these values with the dry copolymer film thicknesses (measured by VASE), the water content of each copolymer film was determined. 2801


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Lubrication Studies. Prior to tribological testing, native and ChABC-degraded articular cartilage samples were glued on the PMMA supports (hemicylinder on plane) of a low-speed tribometer (EMPA Dübendorf, Switzerland). The digested cartilage samples were incubated overnight at room temperature in 0.1 mg/mL PGA-αPMOXA(x)-HBA HEPES II solution. The native cartilage samples used as controls were stored in HEPES II solution. After incubation, all the samples were rinsed with water and mounted on the tribometer. The PGA-PMOXA-HBA-coated samples were left to equilibrate for 15 min in a 0.1 mg/mL PGA-PMOXA-HBA synovial fluid solution before measuring the COF, while for the control samples the equilibration was performed using pure synovial fluid. The measurements were performed in a linear reciprocating approach at 5 mm/s sliding speed, applying two different normal loads in ascending order (5 and 10 N) for 15 min each. Graftcopolymer-coated cartilage samples were prepared in four replicates in order to perform two experiments for each copolymer type. The stability against wear was evaluated at the end of the cycle, restoring the original conditions and repeating the experiment. The COF was calculated as the ratio between the frictional force (averaged between forward and reverse sliding directions) and the applied normal force. Contact area and maximum pressure at contact were calculated using the formulas reported for the Hertzian contact of a hemicylinder on a plane (eq 163), where R and L are the radius and the length of the hemicylinder, respectively, F is the applied normal load, E′ is the contact modulus defined by eq 2, and ν is the Poisson ratio of the cartilage (0.464). Since the sliding speed induces changes in the Young’s modulus (E) of the cartilage,65 two different E moduli were considered for the two cartilage surfaces sliding against each other (E1 = 5 MPa for the sliding sample and E2 = 1 MPa for the fixed sample65).

experiments were performed by implanting the constructs (one copolymer-coated scaffold and one control per animal) subcutaneously in NU/NU nude mice (2−3-month-old female, Charles River). Animal studies were performed in compliance with the ethical guidelines (application number ZH189/2014). After 3 weeks, animals were euthanized via CO2 asphyxiation and the explants fixed for 2 h in 4% paraformaldehyde. Samples were washed, embedded in O.C.T (Tissue-Tek O.C.T Compound Blue, Sysmex), frozen on dry ice, and stored at −80 °C. Sections of 5 μm thick were cut using a Cryostat (CryoStar NX70,Thermo Scientific). The sections were immersed in PBS for 5 min to remove O.C.T. and stained for hematoxylin and eosin (H&E). In addition, the stability of the fluorescent films on the implanted constructs was evaluated by means of a hybrid microplate reader (Synergy H1, BioTek), recording the fluorescein emission spectra both on the scaffolds and on the surrounding skin and comparing them with their duplicates incubated in vitro for the time of the experiment. Cartilage Histological Staining. Articular cartilage punches 2 mm in diameter were removed from knees of 6-month-old bovine calves (Slaughterhouse Zürich) and incubated at 37 °C in the presence of 1 U/mL Ch2905 in 0.01% BSA/PBS solution for 1 h. Similar cartilage cylinders stored in PBS were used as controls. After the enzymatic treatment, the samples were washed three times with 0.01% BSA solution and once with PBS in order to remove the enzyme and fixed in 4% paraformaldehyde for 2 h. Then the samples were washed three times in PBS, embedded in OCT (Tissue-Tek O.C.T. Compound Blue, Sysmex), and snap frozen on dry ice. Sections of 5 μm were cut using a cryostat (CryoStar NX70, Thermo Scientific) and stored at −20 °C until use. The sections were stained with H&E as previously described. Alcian blue staining was carried out by washing the slides with PBS for 5 min, then incubating them in Alcian blue solution (1% in 3% acetic acid, pH 2.5) for 30 min, washed in distilled water, and incubated for 7 min in Nuclear Fast red solution (0.1% in 5% aluminum sulfate). Slides were stained for Safranin-O, Fast green, and hematoxylin by incubating them in Weigert’s iron hematoxylin for 5 min, followed by 0.04% w/v Fast green for 5 min and 2% w/v Safranin-O solution. Before mounting with a resin mounting media, the slides were dehydrated in 95% ethanol. Collagen 2 staining was performed after 30 min of 0.2% w/v hyaluronidase digestion at 37 °C and 1 h of blocking with 5% BSA in PBS with 1:200 diluted rabbit anti-collagen 2 (Rockland 600-401-104). Lubricin staining was performed after 18 min of digestion in 0.05% w/ v trypsin solution containing 1% w/v CaCl2 at 37 °C and 1 h blocking in 5% BSA. Primary rabbit anti-lubricin antibody (ab28484, Abcam plc, Cambridge, UK) was diluted 1:250. Both primary antibodies were diluted in 1% w/v BSA in PBS and incubated overnight at 4 °C. Alexa Fluor 488 goat anti-rabbit (Invitrogen A11008) secondary antibodies were used at 1:200 dilution in 1% BSA in PBS for 1 h at RT. Finally, slides were incubated for 10 min with the nuclear stain DAPI (Molecular Probes, Invitrogen) before mounting with VectaMount AQ mounting medium (Vector Laboratories). Western Blotting on Bovine Synovial Fluid. Standard SDSPAGE electrophoresis of bovine synovial fluid (SF) was performed under denaturing conditions (10 μL/5 μL/2 μL aliquots of denatured SF were loaded in a 4−12% Bis-tris protein gel (NuPAGE, NP0321, Thermo Fisher Scientific, Waltham MA, USA) and transferred to a nitrocellulose membrane (10600001, GE Healthcare, Little Chalfont, United Kingdom). The nitrocellulose membrane was blocked for 1 h in a 5% BSA solution in PBS + 0.05% Tween 20. After washing it with PBS + 0.05% Tween 20, the membrane was incubated overnight at 4 °C in a 1:1000 diluted solution of the primary rabbit anti-lubricin antibody (ab28484, Abcam plc) in PBS + 0.1% Tween 20, 3% BSA, and 0.02% NaN3. Following four washings in PBS + 0.05% Tween 20, the membrane was incubated for 1 h at room temperature in a 1:2000 diluted solution of HRP-linked anti-rabbit secondary antibody in PBS + 0.1% Tween 20 and 3% BSA. Following four more washings in PBS + 0.05% Tween 20, the luminescence signal was recorded on a ChemiDoc (BioRad), using SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific).

A = 2bL = 2

E′ =

8FR L πLE′

(1)

1 − ν2 E2

(2)

2

(

1 − ν2 E1

−

)

The effective contact area was measured applying the tested loads on a glass slide previously coated with fluorescent polystyrene colloids. Under the applied load the colloids at the contact are transferred from the glass slide to the cartilage sample, leaving an empty area on the glass slide, which can be measured using ImageJ (see Figure S12). The calculated and measured values are reported in Table S2 of the Supporting Information.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b07847. Graft-copolymer characterization before (1H NMR) and after (XPS, AFM) chemisorption on APTES-functionalized silicon substrates; in vitro and in vivo experiments; cartilage staining and synovial fluid component evaluation (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Edmondo M. Benetti: 0000-0002-5657-5714 Notes

The authors declare no competing financial interest. 2802


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Nanoassemblies of Tissue-Reactive, Polyoxazoline Graft-Copolymers

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