Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering

International Journal of Biological Macromolecules 103 (2017) 978–989

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering Annalisa La Gatta a,∗ , Giulia Ricci a , Antonietta Stellavato a , Marcella Cammarota a , Rosanna Filosa a , Agata Papa a , Antonella D’Agostino a , Marianna Portaccio a , Ines Delfino b , Mario De Rosa a , Chiara Schiraldi a,∗ a b

Department of Experimental Medicine, School of Medicine, Campania University “Luigi Vanvitelli”, Via L. De Crecchio 7, 80138 Naples, Italy Dipartimento di Scienze Ecologiche e Biologiche, Università della Tuscia, Viterbo, Italy

a r t i c l e

i n f o

Article history: Received 20 December 2016 Received in revised form 30 March 2017 Accepted 16 May 2017 Available online 24 May 2017 Keywords: Hyaluronan Crosslinking Hydrogels Chondrocytes Cartilage engineering

a b s t r a c t In the field of cartilage engineering, continuing efforts have focused on fabricating scaffolds that favor maintenance of the chondrocytic phenotype and matrix formation, in addition to providing a permeable, hydrated, microporous structure and mechanical support. The potential of hyaluronan-based hydrogels has been well established, but the ideal matrix remains to be developed. This study describes the development of hyaluronan sponges-based scaffolds obtained by lysine methyl-ester crosslinking. The reaction conditions are optimized with minimal chemical modifications to obtain materials that closely resemble elements in physiological cellular environments. Three hydrogels with different amounts of crosslinkers were produced that show morphological, water-uptake, mechanical, and stability properties comparable or superior to those of currently available hyaluronanscaffolds, but with significantly fewer hyaluronan modifications. Primary human chondrocytes cultured with the most promising hydrogel were viable and maintained lineage identity for 3 weeks. They also secreted cartilage-specific matrix proteins. These scaffolds represent promising candidates for cartilage engineering. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Currently, the most promising approach for repairing chondral defects with regeneration is tissue engineering that may also combine three-dimensional matrices (scaffolds) with chondrocytes or progenitor cells. The scaffolds must mimic the cartilage microenvironment, and the cells are selected to guide tissue regeneration within the defect [1–6]. The requirements for an ideal scaffold include adequate diffusion of nutrients and metabolites through the matrix (high water content, porous 3D-architecture), proper adhesion to the defect, a suitable degradation rate, and biomechanical properties that resemble, as closely as possible, native cartilage [5,7,8]. An appropriate cell-scaffold interaction is crucial for a successful outcome. The scaffold should adequately support cell attachment, proliferation, and extracellular matrix (ECM) production. Currently, one of the main problems faced in producing the

∗ Corresponding authors. E-mail addresses: [email protected] (A. La Gatta), [email protected] (C. Schiraldi). http://dx.doi.org/10.1016/j.ijbiomac.2017.05.091 0141-8130/© 2017 Elsevier B.V. All rights reserved.

scaffold is how to prevent chondrocyte trans-differentiation into a fibroblastic phenotype, and thus, prevent switching from producing type II to producing type I collagen [7,9]. This control is key in generating a functional tissue with the composition and properties of native cartilage [7,9]. The design of scaffolds that provide biological cues for maintaining the chondrocytic phenotype is, therefore, of great interest. Hyaluronan (HA) is widely used in fabricating matrices for cartilage repair [2,8–16]. A natural component of ECM, HA is known to interact with diverse chondrocyte surface receptors, which positively affect many cellular pathways, including those involved in chondrocyte proliferation, ECM secretion, and phenotype regulation [2,12,17]. However, it is well known that HA must be chemically modified (derivatized or crosslinked) for use in tissue engineering applications. Low degrees of modification are highly desirable, because minimally-modified HA scaffolds closely resemble structures in the natural cellular environment, and thus, they are likely to elicit an appropriate biological response. The HA-based scaffolds proposed to date for cartilage engineering include the commercialized hyaluronan benzyl esters

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(Hyaff products) and methacrylated and thiolated derivatives that undergo photopolymerization and oxidation, respectively, to form the final network [2,9,12–16]. Several studies have reported rather high degrees of substitution at HA carboxylate groups (in the range of 25–100%) [9,13–16]. Furthermore, although interesting results have been achieved, improvements are needed to ensure that the requirements highlighted above are fully addressed. In this study, we aimed to evaluate an alternative HA-based scaffold with a low degree of biopolymer modification for cartilage repair. In particular, we reasoned that scaffolds made of HA crosslinked with lysine methyl-ester (Lys) would provide a highly biocompatible bridging molecule, and we expected it to show high stability in physiological environments, due to the cross-links via amide bonds. Scaffolds were fabricated according to a protocol recently reported by Desiderio et al. (2013), except the reaction conditions were tailored to promote more efficient network formation and a low degree of hyaluronan modification [18]. Thus, several hydrogels were prepared, and each differed by the amount of crosslinker used (range: 1–5% molar ratio of crosslinker to hyaluronan carboxylate). Products were characterized with in vitro chemico-physical analyses to determine swelling and rheological properties, morphology, and stability in cell culture conditions, to verify their suitability for the intended application. The best performing material was then evaluated as a scaffold for culturing primary human chondrocytes in vitro. Cell viability, cell morphology, and the formation of a specific cartilaginous matrix were investigated to ascertain the product’s potential for cartilage regeneration. 2. Experimental section 2.1. Materials Hyaluronic acid sodium salt (HA; Lot. N. 1005062) was purchased from Biophil S.p.a. (Italy). According to suppliers, it is a biofermentative product. The hydrodynamic characterization of the sample was reported elsewhere: it has a weight average molecular weight (Mw ) of 1545 ± 8 kDa, and a polydispersity index (Mw /Mn ) of 1.3 ± 0.1 [19]. We purchased bovine testicular hyaluronidase (BTH, EC 3.2.1.35), essentially a salt-free lyophilized powder with a specific activity of 1877 U/mg (Cat. N. H3884); N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide (EDC); 1-hydroxybenzotriazole hydrate (HOBt), lysine methyl ester (Lys); and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Sigma-Aldrich Chemicals Co. (Milan, Italy). Dulbecco’s phosphate buffered saline (PBS), without calcium and magnesium, was purchased from Lonza Sales Ltd., Switzerland (Cat. N. BE17-512F). Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin (Cat. N. DE17602E), and fungizone (Cat. N. 17836E) were purchased from Life Technologies (Breda, The Netherlands).

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Crosslinking was carried out with carbodiimide chemistry. EDC/HOBt mixtures (1:1 molar ratio) were used to activate HA carboxyl groups (EDC/HOBt:HA molar ratio in the range 10–70%) [18,20,21]. The crosslinker (Lys) was used at 1, 2, and 5% molar ratios, with respect to the hyaluronan carboxylic groups. Reactions in the absence of Lys were also performed as a control. Specifically, the following solutions were prepared: (a) acetone/water (9/1 v/v), which contained 10% EDC, 10% HOBt (10% EDC/HOBt), and varying amounts of Lys (0, 1, 2, or 5%); (b) acetone/water (9/1 v/v); and (c) acetone/water (9/1 v/v), which contained 10% EDC/HOBt. Reactions were started by adding 50 mL of solution (a) to 1.0 g of lyophilized HA. This mixture was slowly stirred at room temperature (about 25 ◦ C) for 20 min. Then, 5mL aliquots of solution (b) or (c) were added every 15 min (2.5 h total reaction time) according to Table 1. Codes for identifying the diverse crosslinked products (XHA) are also shown. Briefly, each XHA product is designated with two values, written as YZ , where Y refers to the mol% Lys used and Z refers to the mol% EDC/HOBt used. Previously, Desiderio et al. (2013) used a higher amount of Lys (30% molar ratio) in the presence of EDC/HOBt (30/30) for scaffold production [18]. It is important to note that HA remained insoluble in the reaction medium during its modification (heterogeneous reaction conditions). A schematic of the crosslinking reactions is shown in Fig. 1. In particular, the active hyaluronan ester that forms in the presence of EDC/HOBt is shown in brackets [20]. The products expected to form in the presence of Lys (Fig. 1, products “a”, “b”, and “c”) and in the absence of the crosslinker (“c”) are labeled XHA. All the XHA products (Table 1) were insoluble in aqueous medium. They were washed in H2 O (650 mL H2 O/g HA, 15 min at 150 rpm), and purification was monitored spectrophotometrically. Purified products were treated with acetone (100 mL acetone/g HA, 15 min at 150 rpm) and dried in a vacuum oven at 40 ◦ C. Samples were weighed, and the (water insoluble) XHA mass recovered was expressed as a percent of the initial HA mass used to initiate the reaction. XHA products were suspended in PBS at a concentration of 6 mg/mL and sterilized in an autoclave (12 min, 120 ◦ C). Sterilized materials are referred to as XHAs materials. The thermal treatment was also intended to hydrolyze ester bonds within the HA networks. Therefore, only “a’“ and “b’“ were expected to resist processing conditions and become the final XHAs products (Fig. 1). After sterilization, the XHAs samples were separated from the liquid phase and washed in highly purified water, until constant conductivity was detected. Then, they were treated with acetone, dried under sterile conditions, weighed, and used for further characterization. The insoluble HA fraction that remained after sterilization was expressed as a percent of the initial HA mass used to initiate the reaction. In addition to the quantitative evaluation of HA insolubility, photographs were captured to monitor the appearance of the materials throughout the process.

2.3. Scaffold evaluation 2.2. Scaffold preparation HA was dissolved in twice-distilled water to a concentration of 3.8% (w/w). This solution (10 g) was placed in a 6-cm diameter tube to form a uniform layer. The layer was lyophilized with an Epsilon 2–6D LSC, version 1.13, freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Germany) to obtain a porous substrate with a high level of pore interconnectivity. The lyophilization program consisted in 3 different phases: Freezing: −20 ◦ C (8 h); 2) Main Drying: −20 ◦ C; 1.030 mbar (1 h); −10 ◦ C, 0.630 mbar (6 h); −5 ◦ C, 0.630 mbar (1 h); 0 ◦ C, 0.520 mbar (2 h); 5 ◦ C, 0.280 mbar (3 h); 15 ◦ C, 0.080 mbar (3 h); 3); Final drying: 20 ◦ C; 0.054 mbar.

2.3.1. Structural characterization with 1 H NMR spectroscopy Unmodified HA and the XHA products were analyzed with 1 H NMR. Samples were prepared as previously described for crosslinked HA materials [22]. Briefly, XHA products were suspended in a HCl solution (pH = 2) at 4 mg/mL and kept at 70 ◦ C, with stirring at 1000 rpm, until no undissolved material could be observed. The solutions obtained were neutralized by adding Na2 HPO4 , then lyophilized. The lyophilized samples were dissolved in D2 O (10 mg/mL) and evaluated with 1 H NMR spectroscopy (conditions: 300 MHz, 64 scans, 80 ◦ C). Spectra were acquired on a Brucker AC 300. All chemical shifts are expressed in terms of ppm.

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Table 1 The diverse stoichiometries applied in this study to optimize crosslinking. The reagents added to 1 g of lyophilized HA are reported on the left. Solutions (a), (b) and (c) are specified in the materials and methods section. The molar ratio (%) of Lys and EDC/HOBt with respect to HA are indicated. The EDC/HOBt was fixed at a 1:1 molar ratio. Thus, the molar% with respect to HA applies to both moieties. The crosslinked products (XHA) reflect the corresponding crosslinking reaction conditions. For example, “210 ” refers to 2 mol% Lys and 10 mol% EDC/HOBt (with respect to HA carboxylates). Reagents added to lyophilized HA (1 g) 6 × 5 mL (b) 6 × 5 mL (c) 6 × 5 mL (b) 2 × 5 mL (c) + 4 × 5 mL (b) 4 × 5 mL (c) + 2 × 5 mL (b) 6 × 5 mL (c) 6 × 5 mL (b) 6 × 5 mL (c) 6 × 5 mL (b) 6 × 5 mL (c)

50 mL (a) Lys 0% 50 mL (a) Lys 1%

50 mL (a) Lys 2% 50 mL (a) Lys 5%

Lys (molar%, with respect to HA carboxylic groups)

EDC/HOBt (fixed 1:1) (molar%, with respect to HA carboxylic groups)

XHA sample designation

0

10 70 10 30 50 70 10 70 10 70

010 070 110 130 150 170 210 270 510 570

1

2 5

Fig. 1. Scheme for scaffold production. (Left) Different amounts of EDC/HOBt are reacted with HA, and different amounts of Lys are added. (Middle) Three products (XHA) are expected in the presence of Lys and EDC/HOBt (a, b, and c), and only one product is expected in the presence of solely EDC/HOBt (c). (Right) After the sterilization process, two products (XHAs ) are expected (a’, b’).

2.3.2. Morphological analyses XHAs 170 , 270 , and 570 (Table 1) were observed with scanning electron microscopy (SEM). Briefly, the samples were swollen at equilibrium in water, frozen, and lyophilized. Then, they were mounted on a stub and coated with gold (Denton Vacuum Desk V) for observation with SEM (Supra 40 ZEISS; EHT = 5.00 kV, WD = 22 mm, detector in lens). 2.3.3. Swelling behavior The swelling behavior of each XHA in highly purified water and PBS was measured gravimetrically, with an analytical balance (Mettler Toledo, XS105 Dual Range). Briefly, XHA samples (170 , 270 , and 570 ) were weighed (dry weight: wd ), immersed in water or PBS (200 mL aqueous medium/g sample), and maintained at 37 ◦ C in a thermostatic bath. After swelling reached equilibrium, samples were withdrawn, blotted with filter paper to remove surface water, and weighed (ws ). The swelling ratio was calculated as follows: Swelling ratio =

Ws Wd

2.3.4. Mechanical characterization Scaffolds were characterized after swelling reached equilibrium in PBS at 37 ◦ C, conditions close to the intended experimental appli-

cation. Measurements were performed with a Physica MCR301 oscillatory rheometer (Anton Paar, Germany) equipped with a parallel plate geometry (plate diameter, 25 mm) and a Peltier temperature control. Profiled plates were used to prevent materials from slipping during measurements. Strain sweep tests were performed, as reported elsewhere, for crosslinked HA hydrogels [22]. Briefly, a strain amplitude (␥) that ranged 0.001-100% was applied during oscillations and maintained at a constant frequency of 1.59 s−1 . The storage modulus (G’) and the loss modulus (G”), and thus the tan ␦, were evaluated. Mechanical spectra were also recorded; G’ and G” were measured as a function of frequency in the range of 0.1–10 Hz, at a constant strain (0.01%), within the linear viscoelastic range.

2.3.5. Stability in cell culture conditions and during enzymatic hydrolysis XHAs products were suspended at 5 mg/mL in DMEM supplemented with 10% FBS, to study stability in cell culture conditions, or in PBS containing 1000 U/mL BTH, to study stability during enzymatic hydrolysis. Samples were incubated at 37 ◦ C with slow stirring for up to 21 days. Material degradation was monitored by acquiring images of the samples at different times of incubation (0, 7, 14, or 21 days).

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Degradation due to BTH action was quantified by following the increase in the wt% of soluble hyaluronan. Briefly, at predetermined time intervals (7 and 21 days), 200-␮L aliquots of supernatant were withdrawn, and replaced with 200 ␮L of BTH solution. The collected samples were adequately diluted, filtered through 0.45-␮m pore filters, and the amount of solubilized hyaluronan content was evaluated with the carbazole assay [23]. The wt% of soluble hyaluronan was calculated as follows: Soluble

HA (%) =

HA mass in solution (mg) × 100 total HA mass (mg)

where “total HA mass” was the initial amount of XHAs added in each condition. 2.4. Biological evaluations with primary human chondrocytes 2.4.1. Chondrocyte isolation and culture Primary cultures of human chondrocytes were prepared from nasal cartilage. Chondrocytes were isolated after 22 h incubation at 37 ◦ C in a humidified atmosphere at 5% CO2 with 3 mg/mL type I collagenase, 4 mg/mL dispase, and 0.5% (v/v) gentamicin. Cultures were expanded in basal media (DMEM with 10% FBS and antibiotics) in cell growth conditions [24]. Cells were detached at 75–80% confluence, then seeded into the scaffolds, as described in the following section. 2.4.2. Cell seeding and culture in 3D scaffolds Weighed, dry samples of XHA570 scaffolds were placed in a 24well plate. A suspension of chondrocytes (about 7 × 105 cell/mL) was carefully added directly onto the material, and swelling was monitored until it reached equilibrium. This resulted in a theoretical cell density of 7 × 105 cells/cm3 for each sample. Cell-laden materials were incubated in cell growth conditions for 30 min; then, cell culture medium (2 mL) was added. The constructs were maintained in cell culture conditions for three weeks, and the medium was changed every three days. 2.4.3. Cell viability At 7, 14, and 21 days of incubation, cell-laden materials were washed twice in PBS, then transferred to a new 24-well or 96well plate. Materials were evaluated for cell viability with MTT and lactate dehydrogenase (LDH) colorimetric assays. The MTT assay was performed as previously described with slight modifications [25,26]. Briefly, 1.5 mL of MTT solution (0.5 mg/mL MTT in DMEM without phenol red, but with 10% [v/v] FBS) was added to each well. After a 3-h incubation, the medium was removed, and 1 mL of 0.1 N HCl in anhydrous isopropanol was added to each well to solubilize the formazan crystals. Samples were placed in the dark at room temperature with stirring to optimize diffusion of the solubilized formazan in the solvent. These suspensions were transferred to 2-mL tubes, centrifuged, and the supernatant was recovered. Supernatant absorbances were measured at 570 nm (A570nm ); the control sample comprised acellular matrices. The resulting A570nm values were normalized with respect to the number of seeded cells and the volume of the recovered sample. The results are plotted vs. the incubation time. The CytoTox96 Non-Radioactive Assay kit (Promega, USA) was used for the LDH assays. This assay quantitatively measures the LDH released upon cell lysis. The samples were combined with 200 ␮L PBS and 10 ␮L of a lysis solution (TRITON X-100) and incubated for 1 h at 37 ◦ C. Subsequently, samples were centrifuged at 9500g for 5 min. Next, 50 ␮L of each lysate was transferred into a 96well plate, combined with 50 ␮L of a reconstituted substrate mix, and incubated for 30 min in the dark at room temperature. Then, 50 ␮L of a stop solution was added to each well, and the result was measured at A490nm in a standard 96-well plate reader; the control

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comprised acellular matrices. The resulting A490 values were normalized with respect to the number of seeded cells and the volume of the recovered sample. The results are plotted vs. incubation time. 2.4.4. Cell morphology analysis with optical microscopy At 7, 14, and 21 days of incubation, the cell-laden XHA570 samples were harvested and fixed in 2.5% glutaraldehyde. The samples were then dehydrated with increasing percentages of ethanol in water, up to absolute ethanol. Next, samples were embedded in epoxy resin, and cut into semi-thin sections (1 ␮m). These sections were stained with toluidine blue and analyzed with bright field optical microscopy. 2.4.5. Immunohistochemical characterization At 7, 14, and 21 days after chondrocyte seeding, the constructs were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, then cut into 15-␮m sections. The samples were then dewaxed, re-hydrated, and rinsed with PBS. Samples were incubated with a primary polyclonal antibody against human type II collagen (a chondrocyte specific marker; 1:40 dilution). Immunolocalization was performed according to the manufacturer’s instructions (Histostain-Plus kit; Zymed Laboratories). The avidin-biotin immunoperoxidase system (with 3,3-diaminobenzedine as chromogen) was used to visualize bound antibodies. The sections were counterstained with hemalum, dehydrated, mounted, and analyzed with bright field optical microscopy. 2.4.6. Two-photon microscopy measurements Cells cultured in XHA570 were observed with two-photon microscopy (TPM). TPM images were obtained with a modified Olympus Fluoview confocal laser scanning head (FV300), coupled to a fs-Titanium:Sapphire (Ti:Sa) laser (Chameleon Ultra, Coherent, Inc. USA), and equipped with an upright Olympus BX51WI microscope. We used a water immersion objective (Olympus XLUMPLANFl20XW, WD: 2 mm, NA: 0.95) for focusing the laser beam and collecting the fluorescence signals from the samples. The laser pulse time width was estimated to be 150 fs with a 76 MHz pulse repetition frequency. For TPM excitation, a 740 nm wavelength was adopted for exciting NADH autofluorescence, and the emission was collected in the 450–550 nm range. The images were acquired with fixed excitation energy, at 10-␮m steps, to depths of up to 200 ␮m below the sample surfaces. All the acquired images were 230 × 230 ␮m2 in area with a resolution of 512 × 512 pixels and a pixel dwell time of 6.4 ␮s. For further details see Delfino et al., 2013 [27]. 2.4.7. Cell ultrastructural analysis with transmission electron microscopy The cell-laden materials were analyzed with transmission electron microscopy (TEM) at 7, 14, and 21 days after seeding. Briefly, samples were harvested and fixed in 2.5% w/v glutaraldehyde. Specimens were postfixed in 1% w/v OsO4, treated with 1% w/v tannic acid, de-hydrated in ethanol, and embedded in epoxy resin. Specimen were cut into ultrathin sections (60 nm), contrasted in an aqueous lead-hydroxide solution, treated with tannic acid, and photographed with a Zeiss-LIBRA120 transmission electron microscope. 2.4.8. Gene expression levels of collagen type II and aggrecan Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression levels of collagen type II (COLIIA1) and aggrecan (AGN) genes in human primary chondrocytes that grew within the scaffold. After 7, 14, and 21 days of incubation, the cell-laden mate® rial was washed with PBS, and 1 mL of Trizol was added. Samples were maintained at room temperature for 15 min, then 200 ␮L chloroform was added, and samples were centrifuged (12000g for

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Fig. 2. (a) Appearance of the HA samples throughout the manufacturing process. (Top row) images of XHA570 (A-E) are representative of all the products obtained with Lys in the reaction; (bottom row) images of XHA070 (A’–E’) are representative of products obtained without Lys in the reaction. (b) Fraction of insoluble HA (wt%) obtained because of chemical modification (XHA; white bars) and the fraction of insoluble HA that resisted sterilization (XHAs ; grey bars). *p < 0.05; ◦ p < 0.01; §p < 0.005.

15 min). The aqueous phase was recovered, and RNA was extracted ® and quantified, according to the Trizol manufacturer’s instructions, as previously described [24,28]. Total RNA was converted to cDNA with a reverse transcription system kit (Promega, Milan, ® Italy). PCR was performed with the iQTM SYBR Green Supermix (Bio-Rad Laboratories s.r.l., Milan, Italy) and primer pairs, described by Stellavato et al. (2016), that specifically recognized COLII and AGN sequences. Results are expressed as the relative expression of specific mRNAs, normalized to the expression of the HPRT housekeeping gene.

2.5. Statistical analysis All reactions were performed at least in triplicate, and each reaction product was analyzed in triplicate; therefore, a minimum of nine samples was analyzed for each reaction. Data are reported as the mean ± SD. A Student t-test was used for comparisons between groups. P-values less than 0.05 were considered significant.

3. Results 3.1. Manufacturing scaffolds and 1 H NMR analyses Fig. 2a shows the appearance of XHA materials throughout the manufacturing process. The XHA570 sample was qualitatively representative of all materials that included Lys in the reaction; the XHA070 sample represented materials that did not include Lys in the reaction. The unmodified HA sponges, cut into different shapes, are shown on the left (A and A’). The next images show the sponges after the reaction, during purification in water (B and B’), and after drying (C and C’). These images clearly show that, even in the absence of Lys, samples were insoluble in aqueous medium and the initial shape was preserved (B and B’). However, sample dimensions were reduced after drying (C and C’). D and D’ show the XHA samples suspended in PBS before sterilization. E and E’ show the same samples after sterilization (XHAs ): no insoluble material was detected for XHA070 (E’); in contrast, in the presence of the crosslinker, samples remained insoluble and retained its initial shape (E). Finally, the dried XHAs sample is shown on the right.

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Fig. 4. SEM images of (A) the lyophilized, unmodified HA sponge and (B-D) representative images of the morphologies observed in the XHAs materials. Scale bar 100 ␮m. Details magnification (2 × ) is also reported. Fig. 3.

1

H NMR spectra for the unmodified HA and the modified XHA570.

Biopolymer insolubility was quantified throughout the process. In particular, the percent of insoluble hyaluronan with respect to the initial amount of polymer (wt%) is shown (Fig. 2b). HA insolubility after reactions (white bars) is a direct consequence of the chemical modification. Each reaction rendered the HA sponge completely water-insoluble (about 100 wt% for all XHA samples). However, the different materials showed diverse resistances to sterilization. Specifically, the XHA0 samples were completely solubilized, but the XHALys materials proved resistant to sterilization, and the degree of resistance depended on the stoichiometry of the reaction. Specifically, for each%Lys, the fraction of insoluble XHAs increased with increasing amounts of EDC/HOBt. For instance, when 1% Lys was used, the insoluble HA fraction increased from about 19–57 (wt%) for EDC/HOBt increases of 10–70%. Moreover, at a fixed concentration of activated carboxylates (e.g., EDC/HOBt 70%), resistance increased with increasing amounts of the crosslinker; for example, the insoluble HA fraction increased from 57 to about 96 (wt%) when Lys was increased from 1 to 5%. Fig. 3 shows a comparison between the 1 H NMR spectra for the unmodified HA and for XHA570 . The HA 1 H NMR spectrum was consistent with data reported in the literature [29]. In particular, the signal located at ␦ = 1.907 ppm was associated with the protons of N COCH3 groups; also, the 2 , 3 , 4 , 5 , and 6 −protons of a HA disaccharide unit were observed at 3.2–3.82 ppm, and anomeric 1 protons were observed at 4.4 ppm. In the XHA570 spectrum, after integrating the peak at ␦ = 2.92 ppm, (CH2 NH) of Lys, we found that Lys was present at 5.2 ± 0.3 mol% (with respect to HA disaccharide). Moreover, the 1 H NMR spectrum showed a methyl singlet from N-methyl protons (2.79 ppm), consistent with the structure of the N-acylurea derivative, which is known to form when EDC chemistry is used to activate the biopolymer carboxylates [30]. Because this by-product was insoluble in aqueous medium, it was not removed in the purification process. Interestingly, when XHA510 material was quantitatively analyzed, Lys appeared to be present at 5.1 ± 0.3 mol% (with respect to the carboxylate moieties). The spectra of XHA170 and XHA270 were qualitatively equivalent, which indicated that Lys was present within the networks; however, in those cases, the signals were too weak to quantify. No variations were observed in the spectra of the samples after sterilization. This analysis was

Fig. 5. Swelling ratio values for the scaffolds immersed in pure water (white bars) and in PBS, pH7.4 (grey bars); *p < 0.05, **p > 0.05.

also performed on XHA3030, which was described by Desiderio et al. (2013). This analysis revealed the presence of Lys in the network at 29.9 ± 0.8 mol% (with respect to hyaluronan). The 170 , 270 , and 570 XHAs scaffolds were selected for further characterization. 3.2. Scaffold morphology, swelling, and mechanical behavior An SEM image of the unmodified sponge (Fig. 4, A) showed a porous structure with open pores, produced with lyophilization. The XHAs scaffolds did not appear significantly different in SEM images. Three other representative images show the morphologies observed with each scaffold (Fig. 4, B-D). Each material exhibited a porous, interconnected 3D architecture, similar to the architecture of the initial sponge, and a wide distribution of pore dimensions, ranging from about 20 to about 180 ␮m in diameter. These samples were swollen in water and lyophilized; then, the images were acquired; thus, their morphology should resemble, to a certain extent, the morphology under physiological application conditions. The swelling ratios, calculated at equilibrium both in water and in PBS, are reported in Fig. 5. When swollen in water, the swelling capacity of the materials decreased with increasing levels of%Lys used in the original reactions. In particular, for increases in the%Lys from 1% to 5%, the swelling ratios decreased from 55 to 32 g/g. For XHA170 and XHA270 , the extent of swelling in PBS was considerably

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longest incubation time tested, XHA270 and XHA570 preserved about 80% and almost 90% of their masses, respectively. The stability of BTH activity during the experiment was also assessed (data not reported).

3.4. Cell viability and morphology of chondrocytes within the scaffold

Fig. 6. (a) Storage modulus (G’) and tan ␦ values for three scaffolds produced with 1–5 mol% Lys, swollen at equilibrium in PBS, and measured in the linear viscoelastic range at 37 ◦ C. (b) Mechanical spectrum for XHA570 measured at 37 ◦ C.

lower than in water. For XHA570 , the swelling capacities in water and PBS were less markedly different, but nevertheless appreciably different (p < 0.05). In contrast, the swelling capacities of diverse materials were not significantly different when immersed in PBS. Amplitude sweep data (Fig. 6a) show values for the storage modulus (G’) and tan delta (G”/G’) of the scaffolds, recorded within the linear viscoelastic range. The storage modulus of the scaffolds increased with the amount of Lys, from about 2 (XHA170 ) to about 10 kPa (XHA570 ). The tan delta values were below one, as expected for covalently crosslinked networks that exhibit elastic behavior (G’ higher than G”). XHA270 and 570 did not differ in tan delta, but the less crosslinked material showed a significantly higher G”/G’ ratio. The mechanical spectrum for XHA570 is shown in Fig. 6b: the G’/G” ratio was substantially maintained over the entire frequency range examined. The same trend was observed for the other samples (data not shown). 3.3. Scaffold stability in cell culture conditions and resistance to enzymatic hydrolysis In the stability studies (Fig. 7), photographs of the samples were taken before (t = 0) and after 7, 14, and 21 days of incubation in cell culture medium or in PBS with BTH 1000 U/mL. The figure shows photographs of the least (XHA170 ) and most (XHA570 ) crosslinked materials. All matrices showed high stability in cell culture medium (Fig. 7a), regardless of the extent of modification. However, in the presence of BTH (Fig. 7b), the material with less crosslinking was solubilized to a greater extent than the material with more crosslinking. Thus, the XHA570 matrix appeared to be more resistant to enzymatic hydrolysis. Similarly, the XHA270 hydrogel was highly stable in the presence of BTH (images not shown). A quantitative analysis of material solubilization in the presence of BTH (Fig. 7c) showed that the degradation rate was inversely related to the%Lys. The mass of the less crosslinked material was reduced to about 60 and 40% of the initial mass at 7 and 21 days of incubation, respectively. Under the same conditions, at the

The MTT and LDH tests (Fig. 8) showed that cells remained viable within the scaffold for up to 21 days of incubation. No significant variation was found in the normalized absorbance values during the experimentation period. Semi-thin sections of XHA570 /chondrocyte constructs were observed with an optical microscope at all the culture times analyzed (Fig. 8b-d). Primary chondrocytes seeded within the scaffold were observable as single cells, as cell aggregates (Fig. 8b), or as more organized spheroid bodies (Fig. 8c-d). The inner parts of spheroid bodies contained extracellular material compatible with ECM components. Spheroid body organization represented the most frequent form of aggregated chondrocytes cultured in this material. This organization seemed to restore, “in vitro”, the isogenic group structure of cartilage tissue. Chondrocytes did not seem to interact with the scaffold tightly, because it was rarely possible to observe cells adhering to the material (Fig. 8b). This observation was consistent with previously published ultrastructural studies, which reported that chondrocytes did not exhibit cellsubstrate adhesive junctions, such as hemi-desmosomes; instead, they only exhibited plasma membrane-substrate adhesive interactions [31–33]. Finally, TPM was used to observe cells seeded within the scaffold throughout the experimentation. TPM provided evidence that the cells were present at different depths, up to 200 ␮m below the surface. Fig. 8e shows a NADH autofluorescence image of a chondrocyte in the XHA570 material at 7 days after seeding. The image shows a cell located at a depth of 100 ␮m. 3.5. Ultrastructural analysis with TEM Fig. 9 shows several electron microscope images that were overlaid to reconstruct one whole cell and its interactions with other cells in a spheroid body. The TEM analysis revealed a highly developed endoplasmic reticulum and a prominent nucleolus. Both structures indicated a biosynthetically active cell. Moreover, ECM fibers and amorphous electron-dense material were clearly observable in the extracellular space, which indicated that the cells had deposited new ECM components (Fig. 9a, c, d, and e). Fig. 9b shows the ultrastructure of a representative, cell-to-cell adhesive interaction observed in cultured cells.

3.6. ECM production analysis with immunohistochemistry and RT-PCR The relative expression levels of COLIIA1 and AGN genes with respect to the HPRT housekeeping gene are shown in Fig. 10a. Quantitative RT-PCR showed that COLIIA1 was expressed constantly in chondrocytes grown within the scaffold throughout the 21-day experiment. Moreover, AGN gene expression increased from 7 to 21 days post-seeding in the scaffold. At the longest time tested, AGN gene expression was about 1.2-fold greater than its expression level at 7 days (p < 0.01). Immunohistochemistry (Fig. 10b, c, d) showed that chondrocytes cultured for 7, 14, and 21 days within XHA570 scaffolds deposited collagen II (reddish-brown staining). The low resolution of the images was due to the relatively thick sections (15 ␮m); how-

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Fig. 7. Scaffold stability. XHAs materials produced with low (XHA170 ) or high (XHA570 ) amounts of Lys were incubated for t = 0 and 7, 14, and 21 days in (a) cell culture medium or (b) PBS with BTH 1000 U/mL. (c) The extent of solubilization of materials after 7-day and 21-day incubations in the presence of the BTH enzyme *p < 0.05.

ever, in our hands, it was not possible to obtain thinner sections with paraffin-embedded samples.

4. Discussion In this study, we aimed to produce slightly modified HA-based scaffolds for cartilage repair by crosslinking the biopolymer (microporous sponges) with lysine methyl ester. Compared to previously reported protocols, we increased the ratio of the carboxylate activating system (EDC/HOBt) to crosslinker (Lys), from 30:30 to (10–70):(1–5) to improve crosslinking efficiency via amide bond formation [18]. We expected this approach to achieve matrices suitable for tissue engineering purposes and reduce the extent of

biopolymer modification, an attractive goal in view of clinical applications. Under the applied conditions, both the amino groups of Lys and the primary hydroxyl groups of hyaluronan can react as nucleophiles (Fig. 1). Consequently, several products are possible. First, amide bonds can form on both ends of the crosslinker to create a bridge of two COO− groups of hyaluronan (Fig. 1 “b”); second, an amide bond may form on one end, which forms a Lys anchor to a single hyaluronan chain (Fig. 1 “a”); and third, no amide might form, but hyaluronan might crosslink via intra- and inter-molecular ester bonds (Fig. 1 “c”). Modifications to hyaluronan were monitored by quantifying the biopolymer insoluble fraction obtained and by performing structural analyses (1 H NMR). These analyses were not reported in

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Fig. 8. Cell viability and morphology within the XHA570 scaffold. (a) Results of the MTT and LDH tests: the normalized absorbance values are shown as a function of incubation time. (b-d) Representative images of semi-thin sections stained with toluidine blue and observed with bright field optical microscopy; chondrocytes were cultured for (b) 7, (c) 14, and (d) 21 days. Black arrows indicate the scaffold. Red arrow indicates the interaction between a cell and the biomaterial. Asterisks indicate newly deposited extracellular matrix. Scale bar applies to images b-d. (e) TPM image shows a cell at a depth of 100 ␮m in the XHA570 scaffold at 7 days after seeding. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Fig. 9. Ultrastructural analysis of chondrocytes within the XHA570 scaffold. (A) The ultrastructure of one whole chondrocyte is reconstructed with different TEM images. White asterisks indicate deposited extracellular matrix; white arrowhead indicates the nucleolus; white arrow indicates the highly developed endoplasmic reticulum. (B-E) The most representative ultrastructural details of chondrocytes within the scaffold. (B) Black arrowheads indicate the adhesive interaction between cultured cells; (C) black arrow indicates rough endoplasmic reticulum cisternae; and (D, E) black asterisks indicate extracellular matrix.

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Fig. 10. Extracellular matrix biosynthesis and deposition. (a) Quantitative gene expression analysis of cartilage markers, COL2A1 and AGN, in human primary chondrocytes after 7, 14, and 21 days of culture within the scaffold. Values are normalized to the level of HPRT housekeeping gene expression (%). *p < 0.01 and ◦ p < 0.001. (b–d) Immunolocalization of collagen II. Immunohistochemical analysis of collagen II in chondrocytes/XHA570 constructs at (b) 7, (c) 14, and (d) 21 days of incubation. Peroxidase conjugated secondary antibody was used to detect immunocomplexes. Brown signal indicates the presence of collagen II. Hematoxylin counterstain was implemented to detect the texture of the scaffold (arrows).

previous studies on XHA materials produced with Lys [18]. The overall results (Fig. 2 and 3) were consistent with the reaction scheme shown in Fig. 1. In particular, the reactions performed in the presence of the activating system, but without Lys, allowed us to distinguish the effects ascribable to amide bond formation from the effects ascribable to autocrosslinking via ester bonds in the final products (Fig. 1). The results for XHA in Fig. 2b indicated that, under the conditions applied, hyaluronan autocrosslinking could make the biopolymer completely insoluble. However, the XHAs products (Fig. 2b) indicated that the ester bonds were insufficient to confer HA resistance to sterilization. This finding supported the description of XHAs materials shown in Fig. 1. Quantitative NMR analyses for XHA510 and XHA570 (Section 3.1) revealed that the crosslinker was completely incorporated into the final network, regardless of the percentage of activated carboxylate moieties. However, XHA570 proved considerably more resistant to solubilization than the other materials, during the thermal treatment (Fig. 2b). When 1% and 2% Lys were used, resistance to solubilization also improved by increasing the amount of EDC/HOBt (Fig. 2b). This suggested that a 2:1 EDC HOBt:Lys ratio (this ratio was 1:1 for XHA3030 ) was sufficient for 100% crosslinker incorporation into the network. Thus, increases in the amounts of EDC/HOBt increased the efficiency of bridge formation, with double-end Lys anchors (product b, Fig. 1) favored over the formation of strings, with single-end Lys anchors (product a, Fig. 1). This indicated that more efficient networks could be formed for a given extent of hyaluronan modification.

The quantitative NMR results also highlighted a key feature of scaffolds production: the degree of biopolymer modification. Indeed, the degree of biopolymer modification in the most highly crosslinked material developed in the present study was 6-fold lower than that reported in scaffolds described previously by Desiderio et al. (2013), and from 2.5 to 10-fold lower than those reported for other hyaluronan-based scaffolds proposed for engineering cartilage [9,13–16,18]. Heterogeneous reaction conditions were another noteworthy aspect of this study. These conditions permitted us to define the scaffold shape (and dimensions) prior to crosslinking (Fig. 2a); thus, we could obtain a final material consistent with the initial shape. This technological aspect could be advantageous in clinical applications, considering the importance of scaffold placement/insertion into the specific defect. Results discussed above indicated that XHA170 , XHA270 , and XHA570 were the “optimized” materials produced with each amount of Lys tested. Therefore, these materials were considered for further characterizations. Specifically, we investigated features like 3D architecture, water uptake capability, mechanical properties, and stability in conditions close to clinical applications to evaluate the suitability of the materials for the intended purpose. The SEM images demonstrated the porosity of the architecture, and upon close examination, pores of the superficial layers appeared to be interconnected. These features are essential for interchange with the culture medium, cell migration, and matrix distribution (Fig. 4). Data in the literature are controversial regarding the most suitable pore size for regenerating cartilage. However,

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in general, pores ranging from about 10 to about 500 ␮m are considered appropriate [13,34–37]. The pore sizes of the XHA scaffolds developed here fell within this range; interestingly, they were larger than the pores reported for other crosslinked HA-based scaffolds [14]. The large pore size can be considered an attractive achievement. Previous studies that compared the same materials at different pore dimensions showed that, with increases in pore size, increases were observed in chondrocyte proliferation and/or ECM production [35,37,38]. The water uptake capabilities of our materials (Fig. 5) were comparable to the capabilities reported for other hyaluronan-based scaffolds proposed for cartilage engineering. The results of the swelling study (Fig. 5) were consistent with the compositions of the materials. When materials were equilibrated in de-ionized water, the extent of swelling decreased with increases in the Lys percentage. This finding was consistent with Lys-associated increases in crosslinking density and decreases in the net charge of the materials [38,39]. Differences in the ability to absorb water became insignificant when materials were equilibrated in PBS (increased ionic strength); this indicated that ionic osmotic pressure was the driving force for water uptake, consistent with the charged nature of these materials [25]. Because the role of cartilage is mainly mechanical, great attention has been focused on the mechanical behavior of newly developed scaffolds. The rheological analyses of the XHAs sponges (Fig. 6a and 6b) revealed values of storage moduli consistent with the degree of crosslinking. Thus, the least modified network exhibited the lowest rigidity, and the network with the highest amount of Lys exhibited the highest rigidity. As expected for hydrogels, the mechanical properties of the XHAs materials did not approach the properties of the native tissue; the storage moduli indicated that the rigidity achieved was about ten-fold lower than that of native tissue. However, notably, these XHAs materials exhibited comparable mechanical behavior to those reported for similar hyaluronan-based materials, but with less biopolymer modification [13,16]. In considering this characteristic, we must also be aware that, based on recent studies, the ideal scaffold must provide a proper balance between its initial rigidity and its permeability. In fact, materials that closely resembled native cartilage, in terms of stiffness, proved to result in non-functional tissue, because they were less permissive in terms of matrix distribution. Conversely, less stiff materials proved to favor the formation of a contiguous matrix thus exhibiting mechanical function that improved over time [14,16,17,40]. The degradation studies (Fig. 7) highlighted the fact that all the materials had similarly sound stabilities in cell culture medium. This finding was consistent with the high resistance of the amide cross-links to chemical hydrolysis under physiological conditions. Sensitivity to hydrolysis catalyzed by BTH was reduced with increases in the degree of crosslinking. However, we observed hydrolyzed hyaluronan chains even in the most crosslinked network, which indicated that they remained recognizable as hyaluronan by the enzyme. Under both conditions, the XHA materials exhibited far greater stability than other hyaluronanbased scaffolds described previously. For example, one report showed that Hyaff materials could not be handled after a few days of incubation in DMEM; also, metacrylated hyaluronan networks produced by Burdick et al. (2009) were completely degraded within 1 day in the presence of a 10-fold lower BTH concentration than the concentration used in the present study [14,41]. Based on the results of the chemico-physical characterizations, XHA570 was considered the most promising material for providing proper support during the initial stages of regeneration. Under physiological conditions, XHA570 retained resorbability, water permeability, and suitable morphology. Our biological studies with primary human chondrocytes demonstrated that the selected material (XHA570 ) provided a

microenvironment that could maintain chondrocyte survival and lineage identity. The MTT and LDH assays indicated that chondrocytes cultured with XHA570 formed a stable population, as expected for fully differentiated chondrocytes, which have a low proliferating index [42,43]. The presence and homogeneous distribution of chondrocytes within the scaffold throughout the experimentation was confirmed with TPM observations conducted at different depths below the surface. TPM imaging was convenient, because it did not require any particular sample preparation that might disrupt the matrix [44,45]. The biochemical and histochemical results showed that chondrocytes maintained a differentiated state, because cultured cells stably expressed collagen II throughout the culture time. Moreover, the immunohistochemical results showed collagen II protein both inside and outside these cells, which confirmed that collagen II was correctly translated and deposited. Ultrastructural studies showed that the newly formed extracellular fibers were small in size and were deposited irregularly, two features of collagen II deposition that are not exhibited by collagen I fibers. Again, this observation confirmed that these cells displayed the lineage identity of hyaline cartilage tissue derivatives. The increase in AGN gene expression over the culture time (Fig. 10a) indicated that, in culture, the XHA enhanced specific cartilage proteoglycan biosynthesis. Again, this finding showed that XHA-associated cultures maintained the chondrocytic phenotype. Additionally, this result gave rise to the intriguing hypothesis that XHA-chondrocyte interactions might enhance gene expression of cartilage-specific proteins that constitute an amorphous ECM. These results strongly indicated that the XHA material was recognized by cells as native hyaluronan. It is noteworthy that cells cultured with XHA maintained all the morphological features of chondrocytes, including the oval shape, isogenic group formation, and weak cell-to-cell and cell-to-substrate interactions. These characteristics were evident with both optical and electron microscopy. 5. Conclusions We successfully optimized conditions for crosslinking hyaluronan sponges with Lys to produce hydrogels intended for cartilage engineering applications. The optimized conditions enabled the production of matrices that outperformed conventional HA-based hydrogels, with less extensive biopolymer modification. The characterization showed that these materials fulfilled the main requirements for scaffolds in cartilage regeneration. Further studies are needed in animal models for better assessments of the translational potential of these newly developed, hyaluronan-based, 3D networks. Acknowledgements The authors gratefully thank Dr. Maria Assunta Frezza, Dr. Claudia Catalano, Dr. Adele Fusco and Dr. Alessia Izzo for their precious technical support. References [1] C. Vinatier, J. Guicheux, Cartilage tissue engineering: from biomaterials and stem cells to osteoarthritis treatments, Ann. Phys. Rehabil. Med. 59 (2016) 139–144. [2] I.L. Kim, R.L. Mauck, J.A. Burdick, Hydrogel design for cartilage tissue engineering: a case study with hyaluronic acid, Biomaterials 32 (2011) 8771–8782. [3] P.A. Levett, F.P.W. Melchels, K. Schrobback, D.W. Hutmacher, J. Malda, T.J. Klein, A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate, Acta Biomater. 10 (2014) 214–223. [4] H. Tan, J. Wu, L. Lao, C. Gao, Gelatin/chitosan/hyaluronan scaffold integrated with PLGA microspheres for cartilage tissue engineering, Acta Biomater. 5 (2009) 328–337.

A. La Gatta et al. / International Journal of Biological Macromolecules 103 (2017) 978–989 [5] H. Ren, C. He, C. Xiao, G. Li, X. Chen, Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering, Biomaterials 51 (2015) 238–249. [6] R. Mardones, C.M. Jofrè, J.J. Minguell, Cell therapy and tissue engineering approaches for cartilage repair and/or regeneration, Int. J. Stem Cells 8 (2005) 48–53. [7] S. Yamane, N. Iwasaki, T. Majima, T. Funakoshi, T. Masuko, K. Harada, A. Minami, K. Monde, S. Nishimura, Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering, Biomaterials 26 (2005) 611–619. [8] F. Khan, S.R. Ahmad, Polysaccharides and their derivatives for versatile tissue engineering application, Macromol. Biosci. 13 (2013) 395–421. [9] P. Brun, G. Abatangelo, M. Radice, V. Zacchi, D. Guidolin, D. Daga Gordini, R. Cortivo, Chondrocyte aggregation and reorganization into three-dimensional scaffolds, J. Biomed. Mater. Res. 46 (1999) 337–446. [10] H. Tan, C.R. Chu, K.A. Payne, K.G. Marra, Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering, Biomaterials 30 (2009) 2499–2506. [11] P.A. Parmar, L.W. Chow, J.P. St-Pierre, C.M. Horejs, Y.Y. Peng, J.A. Werkmeister, J.A.M. Ramshaw, M.M. Stevens, Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration, Biomaterials 54 (2015) 213–225. [12] B.S. C. Chung, J.A. Burdick, Influence of 3D hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis, Tissue Eng. Part A 15 (2009) 243–254. [13] S. Bian, M. He, J. Sui, H. Cai, Y. Sun, J. Liang, Y. Fan, X. Zhang, The self-crosslinking smart hyaluronic acid hydrogels as injectable three-dimensional scaffolds for cells culture, Colloids Surf. B 140 (2016) 392–402. [14] J.A. Burdick, C. Chung, X. Jia, M.A. Randolph, R. Langer, Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid network, Biomacromolecules 6 (2005) 386–391. [15] L.A. Solchaga, J.E. Dennis, V.M. Goldberg, A.I. Caplan, Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage, J. Orthop. Res. 17 (1999) 205–213. [16] I.E. Erickson, S.R. Kestle, K.H. Zellars, M.J. Farrell, M. Kim, J.A. Burdick, R.L. Mauck, High mesenchymal stem cell seeding densities in hyaluronic acid hydrogels produce engineered cartilage with native tissue properties, Acta Biomater. 8 (2012) 3027–3034. [17] M. Leobourg, J.R. Rochina, T. Sousa, J. Mano, J.L.G. Ribelles, Different hyaluronic aced morphology modulates primary articular chondrocyte behavior in hyaluronic acid-coated polycaprolactone scaffolds, J. Biomed. Mater. Res. Part A 101 (2013) 518–527. [18] V. Desiderio, F. De Francesco, C. Schiraldi, A. De Rosa, A. La Gatta, F. Paino, R. d’Aquino, G.A. Ferraro, V. Tirino, G. Papaccio, Human Ng2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid-Lys scaffold fabricate a skeletal muscle tissue, J. Cell. Physiol. 228 (2013) 1762–1773. [19] A. La Gatta, A. Papa, C. Schiraldi, M. De Rosa, Hyaluronan dermal fillers via crosslinking with 1,4-butandiol diglycidyl ether: exploitation of heterogeneous reaction conditions, J. Biomed. Mater. Res. Part B: Appl. Biomater 104 B (2016) 9–18. [20] P. Bulpitt, D. Aeschlimann, New strategy for chemical modification of hyaluronic acid: preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels, J. Biomed. Mater. Res. 47 (1999) 152–169. [21] N.A. Elder, N.M. D’ Angelo, S.C. Kim, N.R. Washburn, Conjugation of ␤-sheet peptides to modify the rheological properties of hyaluronic acid, Biomacromolecules 12 (2011) 2610–2616. [22] A. La Gatta, C. Schiraldi, A. Papa, A. D’Agostino, M. Cammarota, A. De Rosa, M. De Rosa, Hyaluronan scaffolds via diglycidyl ether crosslinking: toward improvements in composition and performance, Carbohydr. Polym. 96 (2013) 536–544. [23] T. Bitter, H.M. Muir, A modified uronic acid carbazole reaction, Anal. Biochem. 4 (1962) 330–334. [24] A. Stellavato, V. Tirino, F. de Novellis, A. Della Vecchia, F. Cinquegrani, M. De Rosa, G. Papaccio, C. Schiraldi, Biotechnological chondroitin a novel glycosamminoglycan with remarkable biological function on human primary chondrocytes, J. Cell. Biochem. 117 (2016) 2158–2169. [25] A. La Gatta, C. Schiraldi, A. D’Agostino, A. Papa, M. De Rosa, Properties of newly-synthesized cationic semi-interpenetrating hydrogels containing

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42]

[43]

[44]

[45]

989

either hyaluronan or chondroitin sulfate in a methacrylic matrix, J. Funct. Biomater. 3 (2012) 225–238. A. La Gatta, A. De Rosa, P. Laurienzo, M. Malinconico, M. De Rosa, C. Schiraldi, A novel injectable poly(epsilon-caprolactone)/calcium sulfate system for bone regeneration: synthesis and characterization, Macromol. Biosci. 5 (2005) 1108–1117. I. Delfino, M. Portaccio, M. De Rosa, M. Lepore, A preliminary investigation on the interaction between sol-gel immobilized glucose oxidase and freely diffusing glucose by means of two-photon microscopy, Proc. SPIE 8588 (2013), 85882U-1-11. A. D’Agostinoo, A. Stellavato, T. Busico, A. Papa, V. Tirino, G. Papaccio, A. La Gatta, M. De Rosa, C. Schiraldi, In vitro analysis of the effects on wound healing of high- and low-molecular weight chains of hyaluronan and their hybrid H-HA/L-HA complexes, BMC Cell Biol. 16 (2015) 19–33. A. D’Agostino, A. Stellavato, L. Corsuto, P. Diana, R. Filosa, A. La Gatta, M. De Rosa, C. Schiraldi, Is molecular size a discriminating factor in hyaluronan interaction with human cells? Carbohydr. Polym. 157 (2017) 21–30. J.J. Young, K.M. Cheng, T.L. Tsou, H.W. Liu, H.J. Wang, Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3, 3-dimethylaminopropyl)carbodiimide, J. Biomater. Sci. Polymer. Edn. 15 (2004) 767–780. T.S. Okada, G. Eguchi, M. Takeichi, The expression of differentiation by chicken lens epithelium in vitro cell culture, Dev. Growth Differ. 13 (1971) 323–336. C.T. Brighton, Y. Sugioka, R.M. Hunt, Cytoplasmic structures of epiphyseal plate chondrocytes. Quantitative evaluation using electron micrographs of rat costochondral junctions with special reference to the fate of hypertrophic cell, J. Bone. Joint Surg. Am. 55 (1973) 771–784. Y. Kato, D. Gospodarowicz, Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor, J. Cell. Biol. 100 (1985) 477–485. S.M. Lien, W.T. Li, T.J. Huang, Genipin-crosslinked gelatin scaffolds for articular cartilage tissue engineering with a novel crosslinking method, Mat. Sci. Eng. C 28 (2008) 36–43. S.M. Lien, L.Y. Ko, T.J. Huang, Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering, Acta Biomater. 5 (2009) 670–679. S. Yamane, N. Iwasaki, Y. Kasahara, K. Harada, T. Majima, K. Monde, S. Nishimura, A. Minami, Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers, J. Biomed. Mater. Res. A 81 (2007) 586–593. D.J. Griffon, M.R. Sedighi, D.V. Schaeffer, J.A. Eurell, A.L. Johnson, Chitosan scaffolds: interconnective pore size and cartilage engineering, Acta Biomater. 2 (2006) 313–320. J.B. Leach, K.A. Bivens, C.N. Collins, C.E. Schmidt, Development of photocrosslinkable hyaluronic acid-polyethylene glycol-peptide composite hydrogels for soft tissue engineering, J. Biomed. Mater. Res. A 70 (2004) 74–82. K.A. Smeds, M.W. Grinstaff, Photocrosslinkable polysaccharides for in situ hydrogel formation, J. Biomed. Mater. Res. 54 (2001) 115–121. I.E. Erickson, A.H. Huang, S. Sengupta, S. Kestle, J.A. Burdick, R.L. Muack, Macromer density influences mesenchymal stem cell chondrogenesis and maturation in photocrosslinked hyaluronic acid hydrogels, Osteoarthritis Cartilage 17 (2009) 1639–1648. E. Milella, E. Brescia, C. Massaro, P.A. Ramires, M.R. Miglietta, V. Fiori, P. Aversa, Physico-chemical properties and degradability of non-woven hyaluronan benzylic esters as tissue engineering scaffolds, Biomaterials 23 (2002) 1053–1063. P.A. Guerne, F. Blanco, A. Kaelin, A. Desgeorges, M. Lotz, Growth factor responsiveness of human articular chondrocytes in aging and development, Arthritis Rheum. 38 (1995) 960–968. G.M. Peretti, E.M. Caruso, I. Papini Zorli, W. Albisetti, Mitotic activity in chondrocyte transplantation from the in vitro phase to the in vivo phase, Chir. Organi. Mov. 85 (2000) 273–280. M. Vielreicher, S. Schurmann, R. Detsch, M.A. Schmidt, A. Buttgereit, A. Boccaccini, O. Friedrich, Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine, J. R. Soc. Interface 10 (2013) 1–21. R. Dittmar, E. Potier, M. van Zandvoort, K. Ito, Assessment of cell viability in three-dimensional scaffolds using cellular auto-fluorescence, Tissue Eng. Part C Methods 18 (2012) 198–204.