Highly Porous Gelatin Reinforced 3D Scaffolds for Articular Cartilage Regeneration.

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Highly Porous Gelatin Reinforced 3D Scaffolds for Articular Cartilage Regeneration Sofia Amadori, Paola Torricelli, Silvia Panzavolta,* Annapaola Parrilli, Milena Fini, Adriana Bigi 3D highly porous (93% total porosity) gelatin scaffolds were prepared according to a novel, simple method, which implies gelatin foaming, gelification, soaking into ethanol and successive freezedrying. Reinforcement of the as-prepared scaffolds (GEL) was performed through immersion in aqueous solutions at different gelatin concentrations. Reinforcement solutions with and without genipin addition allowed to prepare two series of samples:cross-linked and uncross-linked samples, respectively. The amount of gelatin adsorbed onto the reinforced samples increases as a function of gelatin concentration in solution and provokes a drastic improvement of the compressive modulus and collapse strength up to values of about 30 and 4 MPa, respectively. The open and interconnected porosity, although slightly reduced, is still of the order of 80% in the samples reinforced with the highest concentration of gelatin. Water uptake ability evaluated after immersion in PBS for 20 s decreases with gelatin reinforcement. The presence of genipin in cross-linked samples reduces gelatin release and stabilizes the scaffolds in solution. Chondrocytes from human articular cartilage adhere, proliferate, and penetrate into the scaffolds. The evaluation of differentiation markers both on the supernatants of cell culture and by means of quantitative polymerase chain reaction (qPCR) indicates a dose-dependent promotion of cell differentiation.

1. Introduction

S. Amadori, S. Panzavolta, A. Bigi Department of Chemistry ‘‘G. Ciamician’’, University of Bologna, via Selmi 2, 40126 Bologna, Italy E-mail: [email protected] Fax: þ39 051 2099456 P. Torricelli, A. Parrilli, M. Fini Laboratory of Preclinical and Surgical Studies, Research Institute Codivilla Putti – Rizzoli Orthopaedic Institute, via di Barbiano, 40126 Bologna, Italy Macromol. Biosci. 2015, 15, 941–952 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Regenerative medicine needs advanced materials with hierarchical 3D architectures, able to provide a friendly interaction with biological tissues and promote tissue repair. The scaffolds should be designed properly in order to offer a suitable surface chemistry for cell attachment, proliferation, and differentiation, and, once implanted, they must support the tissue regeneration process. To this aim, key parameters are porosity, pore size, and interconnectivity, which must allow cellular migration, as well as nutrient and waste exchange.[1]

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DOI: 10.1002/mabi.201500014

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S. Amadori, P. Torricelli, S. Panzavolta, A. Parrilli, M. Fini, A. Bigi www.mbs-journal.de

Moreover, scaffolds should display structural and chemical properties matching as much as possible those of the extracellular matrix (ECM).[2,3] Collagen type I is the most abundant structural protein of human body, widespread in several tissues, including skin, blood vessels, tendon, cartilage, and bone. It is extensively used for various medical applications, including for fabrication of tissue engineering scaffolds, due to its good biocompatibility, mechanical strength, and enzymatic degradability.[4] Collagen-based porous scaffolds have shown favorable results with regard to chondrocyte adherence and their ability to maintain a differentiated chondrocyte phenotype.[5,6] However, collagen displays antigenicity. At variance, gelatin, which is obtained by thermal denaturation or physical and chemical degradation of collagen, does not express antigenicity. Moreover, it is a completely biodegradable, highly compatible, and cheap raw material, which justifies its successful numerous applications in tissue engineering, drug delivery, wound dressing, and gene therapy.[7–9] Pure and calcium phosphate-enriched gelatin scaffolds stabilized with genipin, a natural crosslinking agent extracted from the fruits of Gardenia jasminoides Ellis, have been demonstrated to be potentially successful candidates for articular cartilage and bone tissue engineering.[9–13] In this study, we developed a new method to prepare genipin cross-linked 3D porous gelatin scaffolds for articular cartilage tissue engineering and we investigated the possibility to modulate the properties of the supports by means of gelatin solution addition to the as-prepared scaffolds. To this aim, we utilized gelatin solutions at different concentrations and analyzed their effect on porosity, water uptake, degradation, and mechanical properties of the scaffolds. Alternatively, gelatin solutions containing genipin were used. Chondrocytes proliferation, activity, and differentiation were determined through in vitro tests. Chondrocytes differentiation markers were evaluated both on the supernatants of cell culture and by means of quantitative polymerase chain reaction (qPCR).

2. Experimental Section Type A gelatin (280 Bloom, Italgelatine SpA) from pig skin was used. Gelatin scaffolds were prepared from a 10% wt/V aqueous gelatin solution. Initially, 15 g of gelatin were dissolved in 130 mL of distilled water at 55 8C and mechanically stirred (600 rpm) for about 5 min to obtain a white foam. Then, 10 mL of an aqueous solution of genipin 0.15% wt/V (Wako, Japan) and 10 mL of a phosphate buffered solution (PBS) 1M at pH 7.4 were added to the foam. After just a few minutes, the foam was deposited on Petri dishes (f ¼ 6 cm) and allowed to gelify at 37 8C for 3 h. During this period of time, the color of the foam changes from white to light blue to dark blue, due to the binding of the genipin with primary

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amino-groups of the gelatin. Then, the samples were washed in 0.1 M glycine aqueous solution for 30 min, and in distilled water for 5 min. Afterwards, the scaffolds were soaked in ethanol for 24 h, then freeze-dried for 24 h at 44 8C and 0.1 mBar. The gelatin scaffolds were labeled GEL.

2.1. Reinforced Samples 2.1.1. Samples Reinforced With Gelatin (Uncross-linked Samples) The samples GEL were enriched with gelatin solutions at different concentrations: 2.5, 5, 10, and 20% wt/V. To this aim, cubic-shaped samples (about 1 1 1 cm, weight ¼ 50 mg) were rinsed into 10 mL of gelatin solution for some minutes. Then, the excess of gelatin was removed from the samples, which were frozen and lyophilized for 24 h at 44 8C and 0.1 mBar. The samples thus reinforced were labeled as follows: G2.5, G5, G10, and G20.

2.1.2. Samples Reinforced With Genipin Containing Gelatin (Cross-linked Samples) In this case, the gelatin solutions at different concentrations, 2.5, 5, 10, and 20% (wt/V), utilized to reinforce the GEL samples, were cross-linked with an aqueous solution of genipin 0.15% wt/V under stirring at 55 8C for 30 min. Cubic-shaped samples (about 1 1 1 cm, weight ¼ 50 mg) were rinsed into 10 mL of crosslinked gelatin solution for some minutes. Then, the excess of gelatin was removed from the samples, which were frozen and lyophilized for 24 h at 44 8C and 0.1 mBar. The samples reinforced with genipin containing gelatin were labeled as follow: G2.5G, G5G, G10G, and G20G.

2.2. Scaffolds Characterization The morphological and microstructural characterizations of the porous composite scaffolds were performed by scanning electron microscopy (SEM) using a Philips XL-20 scanning electron microscope operating at 15 kV. The specimens were sputter-coated with gold before examination. For the determination of the amount of adsorbed gelatin, the samples were weighed before and after the reinforcement process. The amount of adsorbed gelatin was calculated as W% ¼

ðW G W GEL Þ 100 W GEL

ð1Þ

where WGEL is the weight of the sample GEL (50 mg) and WG is the weight of the sample after reinforcement. For each sample, the reported value is the mean of seven determinations and is reported with its standard deviation. To measure the equilibrium Water Uptake Ability (WUA) of the scaffold, pre-weighed dry sample was immersed in PBS for 20 s. After the bulk PBS was removed by placing the wet scaffold on the petri dish for 1 min, the weight of wet sample was

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measured. Then, the water uptake ability was determined according to the following equation: WUA ¼

Ww Wd Wd

ð2Þ

where Ww and Wd represent the weight of wet and dry sample, respectively.[14] The process was repeated with three different specimens of same scaffold and data were reported as mean and standard deviation. Compression tests were performed on 1 1 1 cm samples using a 4465 Instron testing machine, equipped with a 1 kN load cell. The loading rate was 1.0 mm min1. At least six specimens were tested for each sample, and data are reported as mean and standard deviation. Statistical analysis was performed with the Student t-test considering a P value of less than 0.05 to be significantly different. For the determination of gelatin release, each sample (50 mg) was immersed in 5 mL of PBS, pH 7.4, at 37 8C. Fractions were collected and analyzed for gelatin content after 3, 6, 24, 48 h and 7, 14, 21, 28 d. Each sample was analyzed in triplicate. Gelatin concentration in the release buffer was determined by colorimetric assay using a bicinchoninic acid protein assay (Sigma Chemical, St. Louis, MO).[15] A 4% wt/V copper (II) sulfate pentahydrate solution was mixed with an excess of bicinchoninic acid at a final ratio of 1:50 V/V; 200 mL of the release solution were added to 2 mL of the assay solution in a test tube. Following further addition of PBS up to a final volume of 5 mL, the solutions were stored at 37 8C for 30 min, and then cooled to room temperature. The absorbance of each solution at 562 nm was measured using a Cary 50 Bio spectrophotometer. The gelatin concentration in the release solution was determined through comparison with a calibration curve. For quantitative three-dimensional (3D) analysis of the material porosity, the samples were scanned using a high-resolution microCT system SkyScan 1172 (Bruker Micro-CT, Kontich, Belgium). The source voltage and current were set at 20 kV and 110 mA respectively, with a nominal resolution of 6.5 mm (pixel size). The samples were rotated until 1808 with a rotation step of 0.48. The duration of each scan was of nearly 60 min to obtain nearly 1 500 images (2 096 4 000 pixels) in 16-bits TIFF format for each dataset. These dataset were then reconstructed with the software NRecon (version 1.6.8.0, Bruker) to obtain the microtomographic sections (4 000 4 000 pixels, 6.5 mm pixel size) using the specific alignment correction depending on single scan, a smoothing and a ring artifacts reduction. The microtomographic sections were used to create virtual 3D models of the analyzed object that allow realistic visualizations of the samples in space. Quantitative 3D and 2D distribution analyses were carried out using a dedicated software (CTAn v. 1.14.4.1, Bruker) on a cubic volume of interest (VOI) of 5 5 mm. The following morphological parameters were evaluated: (1) total porosity P(tot) (%), expressed as the ratio between the volume of detected pores within the material and the total volume of the VOI; (2) open porosity P(op) (%), expressed as the ratio between the volume of detected open and interconnected pores within the material and the total volume of the VOI;


(3) closed porosity P(cl) (%), expressed as the ratio between the volume of detected closed and not interconnected pores within the material and the total volume of the VOI. Distribution of total porosity P(tot)Ai (%), expressed as the ratio between the total area of pores within the material detected in every i section of the VOI and the total area of the section i, extended for 5 mm (height of the VOI).

2.3. Cell Cultures A normal human primary chondrocyte culture derived from the human knee articular cartilage (NHAC-kn, CloneticsTM Cell System, Lonza Milano srl, BG, I), was used for the experiment. Cells were expanded in monolayer cultures, using Chondrocyte Growth Medium (CGM, containing FBS 5%, gentamicin sulfateamphotericin B 0.1%, bFGF-B 0.5%, R3-IGF-1 0.2%, insulin 0.2%, transferrin 0.1%). When NHAC reached 70–80% confluence, cells were detached from culture flasks by trypsinization, and centrifuged; cell number and viability were checked with Trypan Blue dye exclusion test (Sigma, UK). A cell suspension of 2.5 105 cells mL1 at the first passage was used for experiment. Chondrocytes were seeded as pellet of concentrated cells on six sterile samples for each of the following materials: GEL, G2.5, G5, G10, G2.5G, G5G, and G10G. Before cell seeding, the samples were sterilized with g- rays (25 kGy).[13,16] The same concentration of cells was also seeded on polystyrene of the culture plate as control (CTR). A differentiating medium to activate chondrocytes (Chondrocyte Differentiation Medium – CDM – supplemented with TGFb-1 0.5%, R3-IGF-1 0.2%, insulin 0.2%, transferrin 0.2% and ascorbic acid 2.5%) was used. Cultures were maintained at standard condition at 37 8C 0.5 with 95% humidity and 5% CO2 0.2 up to 14 d.

2.4. Cell Proliferation, Viability, and Activity At 7 and at 14 d, WST1 test was performed to assess cell proliferation and viability. 100 mL of WST1 (tetrazolium salt) and 900 mL of fresh culture medium were added at every well and cultures were incubated at 37 8C for further 4 h. Tetrazolium salt is transformed to formazan by reductase of mitochondrial respiratory chain, active in viable cells only. Supernatants were measured by spectrophotometer at 450/625 nm. Results were reported as optical density (OD) and values directly correlate with cell number. Supernatant was collected at 7 and at 14 d for the evaluation of most common markers of chondrocyte differentiation: COLL2 (Collagen type II, USCN Life Science, China), AGC (Aggrecan, USCN Life Science, China), and CTSB (Cathepsin B, Boster, CA, USA).

2.5. Cell Morphology Samples for each material, at the end of the experiment times, were processed for Scanning Electron Microscopy (SEM): osteoblasts grown on the materials were fixed in 2.5% (wt/V) glutaraldehyde, in pH 7.4 phosphate buffer 0.01M for 1 h and dehydrated in a graded


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Table 1. Analyzed genes description.

Sequence (50 to 30 )

Gene GAPDHa SOX9

a

FW RV FW RV

TGG TAT CGT GGA AGG ACT CA GCA GGG ATG ATG TTC TGG A GAG CAG ACG CAC ATC TC CCT GGG ATT GCC CCG A

Number of cycles

568C

25

608C

45

reference gene.

ethanol series. After a passage in hexamethyldisilazane, the samples were air dried. The samples were sputter-coated with Pd prior to examination with a Philips CM100 Scanning Electron Microscope.

2.6. Quantitative Polymerase Chain Reaction (qPCR) Total RNA was prepared from all experimental samples and CTR after 7 and 14 d of culture. Phenol-chloroform extraction was performed using TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA was precipitated with isopropanol, washed with ethanol 75%, and then subjected to a step of clean-up with sodium acetate to eliminate organic residues. RNA was reverse transcribed with Superscript VILO cDNA Synthesis kit (Invitrogen, Carlsbad, CA), following manufacturer’s instructions. The resulting cDNA from each sample was quantified with Quant-iT Pico-Green dsDNA assay kit (Invitrogen) and diluted to the concentration of 5 ng mL, in order to exploit the same range of amplification efficiency and to standardize the amount of starting cDNA. qPCR analysis was performed in a LightCycler Instrument (Roche Diagnostics GmbH, Mannheim, Germany) using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) following the ensuing protocol: initial denaturation at 94 8C for 150 , amplification (35 cycles: 94 8C 1500 , appropriate annealing temperature for each target as detailed in Table 1 for 2000 and 72 8C for 2000 ). The protocol was concluded by melting curve analysis to check for amplicon specificity. Two microliters (10 ng) of each sample in duplicate was processed for genes under study. The progressive accumulation of PCR products was monitored at each amplification cycle by measuring the increase in fluorescence due to the binding of SYBR Green I Dye to dsDNA. The crossing point values (i.e., the cycle number at which the detected fluorescence exceeded the threshold value) were determined for each sample, and these values were used for comparative gene expression analysis employing the 2DDCt method. Statistical analysis. Statistical evaluation of data was performed using the software package SPSS/PCþStatisticsTM 21 (SPSS Inc., Chicago, IL, USA). The study is the results of three independent experiments and data are reported as mean standard deviations (SD) at a significance level of P < 0.05. After having verified normal distribution and homogeneity of variance, a one-way ANOVA was done for comparison between groups. Finally, post hoc multiple comparison test (Dunnett) was performed to detect significant differences among groups and controls. Student’s t-test was used for the comparison between two groups.

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Annealing temperature

3. Results and Discussion 3.1. Scaffolds Characterization The novel approach developed to prepare gelatin scaffolds, which implies foaming, gelification, soaking into ethanol, and successive freeze-drying (Scheme 1) provides highly porous 3D materials, as shown by the cross-sectional SEM images which show a porous interconnected structure (Figure 1a).

Scheme 1. Synthesis of reinforced scaffolds.




Figure 1. SEM micrographs of (a) GEL, (b) G5 and (c) G20 scaffolds. Bars ¼ 200 mm.

Reinforcement with gelatin provokes a slight reduction of porosity, as results from the comparison of the morphology of GEL sample with G5 and G20 (Figure 1a–c). Even when reinforcement is carried out with genipin containing gelatin (cross-linked samples), the scaffolds


retain a high porosity, although it seems to undergo a modest reduction on increasing gelatin concentration in the reinforcement solution (Figure 2a–d). In particular, both G10G and G20G seem to exhibit a greater amount of closed cells with respect to G2.5G and G5G. However, the porosity of the cross-linked samples is still very high and completely open as shown by the results of micro-CT analysis. 3D reconstructed images of some scaffolds are reported in Figure 3. The calculated porosity parameters reported in Table 2 indicate that the porosity of the analyzed materials is exclusively due to open and interconnected porosity. A reduction in total porosity P(tot) has been detected in 3D analysis by increasing the percentage of reinforcement both with gelatin and crosslinked gelatin. The 2D distribution analysis show that the total porosity distribution (P(tot)Ai) is homogeneous along the thickness of uncross-linked scaffolds reinforced with low concentrations of gelatin, whereas the porosity of samples G10 and G20 appears slightly higher in the central region of the scaffold. At variance, cross-linked samples display a homogeneous distribution of porosity along the whole thickness of the scaffold even when gelatin concentration is relatively high. Figure 4 shows the total porosity distribution along the thickness of the scaffold (height of the volume of interest, VOI) for some selected samples. The amount of gelatin adsorbed onto the reinforced samples increases as a function of gelatin concentration in solution, provoking an increase of the weight of the scaffold up to about 250% (Figure 5). The data indicate that the average amount of gelatin taken up by the scaffold corresponds to about 10% of the biopolymer content in the 10 mL soaking solution both for uncross-linked and cross-linked samples. The quantity is slightly less for G20 e G20G, most likely because of the greater viscosity of the solution at relatively high gelatin concentration. Reinforcement induces a significant decrease of the water uptake ability: the value obtained for G2.5 (1363 77) corresponds to about 30% of that of the control GEL scaffold (4318 88), and the WUA keeps on decreasing on increasing gelatin content of the reinforced scaffolds down to 575 195 (about 13%), although the difference in WUA between G10 and G20 are not statistically different (P > 0.2), in agreement with the porosity data (Table 2). Cross-linked reinforced scaffold exhibit slightly smaller values of WUA, although the differences with respect to uncross-linked samples are not significant (Figure 6). Moreover, gelatin addition provokes a remarkable improvement of the mechanical properties of the scaffolds. The compressive stress–strain curves of the scaffolds, which show distinct linear elastic, collapse plateau, and densification regimes,[13,17] have been used to evaluate the


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Figure 2. SEM micrographs of (a) G2.5G, (b) G5G, (c) G10G and (d) G20G scaffolds. Bars ¼ 200 mm.

Figure 3. 3D m-CT representation of (a) GEL, (b) G2.5G, (c) G5G and (d) G10G scaffolds. Bars ¼ 500 mm.

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Figure 4. Total porosity distribution – P(tot)Ai – of the scaffolds along the VOI height with the related standard deviation. The values are expressed as the total percentage of pores area detected in every section of the VOI.

mechanical parameters reported in Table 3. The poor mechanical properties of GEL sample are justified by its high porosity. However, reinforcement with gelatin induces a significant increase of the values of the mechanical parameters without dramatically affecting porosity, which retains very high values even in crosslinked samples. Both the linear elastic modulus (E), determined via linear regression of the initial linear regime, and the collapse stress (s), evaluated at the point of transition from linear to collapse regime, increase significantly on increasing gelatin content from GEL to G10, whereas the differences between the values calculated for G10 and G20 are not statistically significant. On the other hand, the slope of the collapse plateau (Ds/De), determined via linear regression of the collapse plateau regime, increases with gelatin content up to G20. The values of strain (e) do not exhibit significant variation with composition. Similar trends of E, s, e, and Ds/De have been observed for cross-linked samples, which

Figure 5. Weight increase of reinforced samples.

exhibit mechanical parameters similar to their analogous uncross-linked samples. The improvement of mechanical properties in compression caused by gelatin reinforcement yields E and s values much greater than those reported for gelatin and/or collagen-based porous scaffolds[18–22] and even greater than those reported for scaffolds containing significant amounts of inorganic phase.[12,13,23–25] These data indicate that the approach used in this work allows to prepare scaffolds containing just one biomimetic material, gelatin, with tailored mechanical properties, and suggest that modulation of gelatin reinforcement could be utilized to develop suitable scaffolds not just for articular cartilage[26] but also for osteochondral applications.[27] The results of the analysis of cumulative gelatin release in PBS reported in Table 4 indicate that gelatin loaded onto the scaffolds is released faster than that of the as-prepared scaffold. In fact, GEL samples show a very small release up to 48 h, after which it increases up to about 23 wt% at 21 d. At variance, the amount released from reinforced scaffolds

Table 2. Porosity parameters of the different scaffolds.

Sample GEL G2.5 G5 G10 G20 G2.5G G5G G10G G20G

P(tot)

P(op)

P(cl)

93.44 0.16 84.27 0.18 78.43 0.37 81.51 0.71 80.30 2.19 84.08 0.16 82.76 3.05 79.21 0.33 80.10 0.71

93.44 0.16 84.27 0.18 78.43 0.37 81.50 0.71 80.14 2.35 84.08 0.16 82.76 3.05 79.21 0.33 80.10 0.71

0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.01 0.72 0.75 0.00 0.00 0.00 0.00 0.02 0.01 0.02 0.01


Figure 6. Water uptake ability of the different scaffolds.


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Table 3. Mean values of linear elastic modulus (E), collapse stress (s) and strain (e), and slope of the collapse plateau (Ds/De) of the 3D scaffolds. Each value is the mean of 6 determinations and is reported with its standard deviation. (Student’ t-test: aP < 0.005, b P < 0.001, cP < 0.0005, dP < 0.0001). s [MPa]

Sample

E [MPa]

e [%]

Ds/De [MPa]

GEL G2.5 G5 G10 G20 G2.5G G5G G10G

0.12 0.03a,c, 0.4 0.1c,d 0.7 0.1a,d 1.9 0.6 3.7 0.8 0.3 0.1a,c,d 0.5 0.1c,d 2.3 0.7

d

1.1 0.4a,b,c, 6 2a,c 8 3a 15 3 24 5 3.3 0.3a,c 6 2a,b 28 8

d

12 1 0.05 0.02c,d 9 2 0.6 0.2a,d 11 3 1.5 0.2a,d 13 3 2.8 0.5c 15 2 71 11 2 0.3 0.1b,c 10 2 0.8 0.1b 12 3 62

E: aG2.5 versus G10; G5 versus G20 ; GEL versus G20G; G2.5G versus G5G, G20G; G5G versus G20G. bGEL versus G2.5G, G5G; G5G versus G10G. cGEL versus G5, G10G; G2.5 versus G20; G2.5G versus G10G. d GEL versus G10, G20. s: aG5 versus G10; GEL versus G2.5G; G2.5G versus G5G. cG2.5 versus G10; GEL versus G20G; G2.5G versus G20G; G5G versus G20G. dGEL versus G2.5, G5, G10, G20, G5G, G10G; G2.5 versus G20; G5 versus G20; G2.5G versus G10G; G5G versus G10G. Ds/De: aG2.5 versus G5; G5 versus G10. bG2.5G versus G5G; G5G versus G10G, G20G. cGEL versus G2.5, G10G, G20G; G10 versus G20; G2.5G versus G10G, G20G. dGEL versus G5, G10, G20, G5G; G2.5 versus G10, G20; G5 versus G20.

increases as a function of composition and reaches values up to about 40 wt% already at 48 h. Prolongation of immersion time up to 28 d resulted in complete dissolution of GEL and of all the uncross-linked samples. On the contrary, gelatin cumulative release from crosslinked reinforced scaffold appears significantly reduced: the values amount to about 10 wt% after 48 h in PBS and increase with time up to 30–39 wt% after 28 d, displaying a

much greater stability in solution of the cross-linked samples with respect to uncross-linked samples. 3.2. In Vitro Biological Tests On the basis of the above reported results, which indicate similar properties for the scaffolds reinforced with solutions at gelatin concentration of 10 and 20%, biological tests were carried out on G2.5, G5, G10, G2.5G, G5G, G10G and on GEL as a reference material. To investigate the behavior of chondrocytes cultured on the scaffolds, concentrated suspension of chondrocytes from human knee articular cartilage, NHAC, was cultured in differentiating medium up to 14 d on the different samples. Viability and activity of NHAC were evaluated after 7 and 14 d of culture. Cell proliferation assessed by WST1 test (Figure 7a), at 7 d was regular, and viability was over 80% in all group in comparison with CTR. Cell number significantly increased in all groups (P < 0.0005) from 7 to 14 d. In particular, G10 and G10G groups showed a slight but significant lower proliferation when compared to CTR, G2.5, G5, G2.5G (7 d) and CTR, G2.5G (14 d). These data assess that gelatin scaffolds reinforced with gelatin both without and with genipin are able to support cell growth without causing any cytotoxity. This is also confirmed by SEM analysis of materials after 14 d of cell culture: chondrocytes attached and spread on all the samples, they displayed numerous filopodia extensions and covered not just the surface but also penetrated into the pores of the scaffolds, as shown in Figure 8, for a series of samples at the end of experimental time. A suitable scaffold for cartilage tissue engineering should not only support cell proliferation, but it must promote and maintain chondrocytic phenotype, i.e., differentiated cells capable of regenerating a physiologically functional cartilage matrix. Collagen type II and Aggrecan were chosen as common markers of differentiated

Table 4. Gelatin release (wt%) from the different samples as a function of the storage time in PBS. Each value is the mean of three determinations and is reported with its standard deviation.

Gelatin release (wt%) Samples GEL G2.5 G5 G10 G20 G2.5G G5G G10G G20G

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t¼3h

t¼6h

t ¼ 24 h

t ¼ 48 h

t¼7d

t ¼ 14 d

t ¼ 21 d

t ¼ 28 d

4.4 0.3 15 2 21.9 0.4 14 2 19 3 7.9 0.1 7.3 0.2 4.5 0.2 4.1 0.1

4.8 0.2 20.9 0.9 28.7 0.1 38 2 38 1 9.3 0.1 8.2 0.1 5.7 0.2 6.5 0.3

5.6 0.7 22.2 0.2 29.8 0.1 40.1 0.6 40.5 0.2 10.3 0.1 9.5 0.1 81 10.5 0.8

6.03 0.01 22.3 0.2 29.8 0.1 40.3 0.3 40.5 0.2 10.3 0.1 9.9 0.1 9.2 0.1 12.6 0.3

8.8 0.1 24.7 0.6 31.0 0.1 40.8 0.1 41.7 0.1 15.9 0.1 20.5 0.4 16.2 0.3 23 1

13 1 27.7 0.8 32.9 0.1 42.8 0.1 44 1 24.0 0.7 31.7 0.2 24.1 0.3 31.4 0.4

23 2 34 2 35.4 0.7 44.5 0.4 45.7 0.3 27.6 0.8 36.8 0.4 30.3 0.7 36.0 0.5

– – – – – 30 1 39.3 0.3 35.5 0.2 39.5 0.6




Figure 7. (a–d). Chondrocytes proliferation (WST1, a) and activity (COLL2, b; AGC, c) after 7 and 14 d of culture on GEL with different percentage. (*P < 0.05; **P < 0.005; ***P < 0.0001). (a) WST1. 7 d: G10 and G10G versus CTR ***; G2.5**; G5**; G2.5G*. 14 d: CTR versus GEL*; G2.5*; G5*; G10**; G5G*; G10G* ; G2.5G versus G10*; G10G*. (b) COLL2. 7 d: CTR versus G10**; G2.5G*; G10G*; GEL versus G2.5*; G5*; G10**; G2.5G**; G5G*; G10G**; G2.5, G2.5G, G5G versus G10*. 14 d: G5, G5G versus G2.5G*; G10G*. (c) AGC. 7 d: G2.5 versus CTR***; GEL**; G10**; G2.5G*; G5G**; G10G**; G5 versus CTR**; GEL**; G10**; G5*; G5G*; G2.5G versus CTR**; GEL**; G10*; G10G*; G5G, G10G versus CTR*. 14 d: G2.5 versus CTR*; GEL*; G2.5G**; G5G*. (d) CTSB. 7 versus 14 d, *all groups.



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chondrocytes to evaluate differentiation and activity of seeded chondrocytes. The extracellular matrix produced by chondrocytes is mainly composed by collagen and proteoglycans. Among them, COLL2 and AGC are the major components, respectively.[28] COLL2 (Figure 7b) at 7 d was significantly higher in CTR group when compared to G10, G2.5G, G10G, and in GEL group when compared to G2.5, G5, G10, G2.5G, G5G, G10G. Among both cross-linked and uncross-linked materials, 2.5 and 5% concentration showed significant higher values then other experimental groups. At 14 d, the differences among CTR, GEL, and experimental groups were no longer significant; however, G5 and G5G maintained the highest significant values in comparison to G2.5G and G10G (P < 0.05).

The amount of AGC at 7 d was significantly higher in G2.5, G5, and G2.5G in comparison to CTR and GEL, and in G5G, G10G in comparison to CTR. At 14 d, G2.5 group was significantly higher than CTR, GEL, G2.5G, and G5G groups. Data about chondrocytes activity related to the production of COLL2 and AGC showed that the studied reinforced scaffolds are able to stimulate and maintain chondrocytes differentiation. In fact, both proteins play an important role in structure and function of cartilage. To better define the maintenance of the differentiated state of chondrocytes, also CTSB, a not common marker of chondrocytes differentiation, has been evaluated. In fact, it has been reported that undifferentiated cells produce high amount of this peptidase, whereas its production decreases

Figure 8. SEM micrographs of chondrocytes on (a) GEL, (b) G2.5, (c) G5, (d) G10, (e) G2.5G, and (f) G10G at 14 d. Bars ¼ 10 mm.

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Figure 9. SOX9 gene expression related to chondrocyte differentiation during the in vitro culture.

during cell differentiation.[29] In our study, the production of CTSB was significantly reduced from 7 to 14 d (P < 0.05), as a further confirmation of chondrocytes in vitro differentiated state. Finally, the gene expression of SOX9 was analyzed after 7 and 14 d of culture. SOX9 is a transcription factor that plays a key role in the processes of chondrogenesis and its activation is required for chondrocytes differentiation.[30] Results of experimental groups were compared to CTR values at 7 and 14 d (Figure 9). Even if the level of SOX9 reached values higher than CTR at different times (7 or 14 d), upregulation of gene expression confirmed that cells of all groups were well differentiated.

4. Conclusion The simple reinforcement procedure developed in this study provides highly porous 3D gelatin scaffolds with tailored mechanical properties and open and interconnected porosity. The presence of genipin in the reinforcement solutions stabilizes the scaffolds against dissolution in PBS solution. The results of in vitro tests indicate that human chondrocytes display good adhesion, proliferation, and penetration into the scaffolds. The scaffolds reinforced with 2.5 and 5% gelatin display the highest values of differentiation markers. However, all the samples promote differentiation. The good cell response, high porosity, and excellent mechanical properties suggest a good performance of these scaffolds for articular cartilage regeneration and allow to hypothesize possible developments for osteochondral applications.

Acknowledgement: This work was financially supported by MIUR (FIRB n8RBAP10MLK7).

Received: January 18, 2015; Revised: March 3, 2015; Published online: March 19, 2015; DOI: 10.1002/mabi.201500014


Keywords: cartilage regeneration; chondrocyte culture; gelatin scaffolds; high-resolution micro-CT; mechanical characterization

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