Elsevier

Applied Surface Science

Volume 403, 1 May 2017, Pages 112-125
Applied Surface Science

Full Length Article
Chondroitin sulfate immobilization at the surface of electrospun nanofiber meshes for cartilage tissue regeneration approaches

https://doi.org/10.1016/j.apsusc.2016.12.135Get rights and content

Highlights

  • Chemical immobilization of chondroitin sulfate at the surface of nanofiber meshes.

  • CS-immobilized NFMs showed lower roughness and higher hydrophilicity.

  • CS-immobilized NFMs offer a highly effective substrate for hACs phenotypic stability.

Abstract

Aiming at improving the biocompatibility of biomaterial scaffolds, surface modification presents a way to preserve their mechanical properties and to improve the surface bioactivity. In this work, chondroitin sulfate (CS) was immobilized at the surface of electrospun poly(caprolactone) nanofiber meshes (PCL NFMs), previously functionalized by UV/O3 exposure and aminolysis. Contact angle, SEM, optical profilometry, FTIR, X-ray photoelectron spectroscopy techniques confirmed the success of CS-immobilization in PCL NFMs. Furthermore, CS-immobilized PCL NFMs showed lower roughness and higher hydrophilicity than the samples without CS. Human articular chondrocytes (hACs) were cultured on electrospun PCL NFMs with or without CS immobilization. It was observed that hACs proliferated through the entire time course of the experiment in both types of nanofibrous scaffolds, as well as for the production of glycosaminoglycans. Quantitative-PCR results demonstrated over-expression of cartilage-related genes such as Aggrecan, Collagen type II, COMP and Sox9 on both types of nanofibrous scaffolds. Morphological observations from SEM and LSCM revealed that hACs maintained their characteristic round shape and cellular agglomeration exclusively on PCL NFMs with CS immobilization. In conclusion, CS immobilization at the surface of PCL NFMs was achieved successfully and provides a valid platform enabling further surface functionalization methods in scaffolds to be developed for cartilage tissue engineering.

Introduction

Cartilage tissue contains large amounts of extracellular matrix (ECM) [1]. The ECM is a complex hierarchical fibrous structure of proteins and polysaccharides such as collagen, proteoglycans, glycosaminoglycans (GAGs) and elastin [2]. These ECM components are continuously synthesized, secreted, targeted and modified by the adjacent cells [3]. In their structural role, GAGs are pivotal in cluster complexes and protein-protein interactions on the cell surface or in the ECM and, consequently, they are involved in signaling or inhibiting cellular events. As a result, GAGs are empowered to influence cell adhesion, migration, proliferation and differentiation [4]. Thus, the ECM components are crucial to the cartilaginous tissue and to the generation of neocartilage.

A large number of biodegradable macromolecules have been developed as temporary synthetic ECMs analogues for cartilage tissue engineering [1], [5], [6], [7]. Specifically, chondroitin sulfate (CS), a type of GAG, is an important constituent of cartilage ECM, contributing to its outstanding properties [8]. CS is involved in the aggregation of large proteoglycans such as aggrecan [9]. The high negative charge of CS creates a charge gradient that swells the cartilage and enhances the tissués ability to sustain loads [10]. Therefore, a number of recent studies have reported on the potential application of CS as a biomaterial for tissue engineering strategies [7], [11], [12], [13], [14], [15], [16], [17].

Polymeric fibrous scaffolds prepared by electrospinning technique have been the subject of extensive research efforts. Electrospun scaffolds have a fibrous morphology which can mimic the morphology of natural ECM of most connective tissues [2]. However, electrospinning of chondroitin sulfate is rather difficult due to the low viscosity of the aqueous polymeric solution, since CS is an extremely water-soluble polysaccharide [18]. Therefore, the low stability in solution and the low mechanical strength of the non-crosslinked CS fibers hinder the use of this material on tissue engineering strategies.

Biomaterials surface properties play important roles in the regulation of cellular behavior such as adhesion, proliferation and differentiation. Indeed, tailoring the surface properties of biomaterials to achieve a specific biological response became one of the central questions in biomedical materials and tissue engineering. Hence, the cytocompatibility of biomaterials can be obtained without altering their bulk properties by using surface coatings or surface modifications. This strategy represents a way to preserve the mechanical properties of the established materials and to improve their biocompatibility [19]. During the past decade, many researchers have studied different surface modification strategies to improve the cell adhesion on synthetic materials including laser treatment [20], plasma treatment [21], ultraviolet (UV) irradiation [22], [23], electrostatic assembly [24], [25] or covalent bonding [7], [26], [27]. Among the different surface functionalization strategies it can be highlighted the coating of the scaffolds surface by several kinds of natural macromolecules including fibronectin [28], [29], gelatin [2], [23], [30], collagen [31], [32], chitosan [33], laminin [34], peptides [22], [35] and chondroitin sulfate [6], [25]. The resulting biofunctional scaffolds have facilitated cell adhesion, proliferation and differentiation of different cell types.

The functionality of electrospun fibers was enhanced by surface modifications similar to the methods previously described [19], [36], [37], [38]. Indeed, electrospun polycaprolactone (PCL) nanofiber meshes were previously proposed as scaffolds for the regeneration of cartilage [5], [39], [40], skin [41] and bone [42]. We herein modified electrospun PCL nanofiber meshes (NFMs) with a simple technique that was developed to introduce free amino groups onto polyester surfaces by aminolyzing the ester groups with diamine molecules [7], [26], [29], [43], [44]. To the best of our knowledge, the chemical immobilization of chondroitin sulfate on electrospun nanofiber meshes was not reported in literature. The presence of different types of chemical groups facilitates the CS immobilization by covalent bonds [6], [45] or by physical interactions [46]. Thus, the aim of this work is the immobilization of chondroitin sulfate on the surface of electrospun PCL NFMs to improve its biological performance for cartilage regeneration, with an optimized surface chemistry for cell recognition/adhesion and signaling.

Section snippets

Reagents

Polycaprolactone (PCL, Aldrich) with a Mn 70,000–90,000. Chondroitin sulfate (CS), kindly supplied by Solabia (Maringá, Brazil), with a MV: 22 × 103 g mol−1, according to Wasteson [47]. Chloroform (Aldrich), Dimethylformamide (DMF, Aldrich), 1,6-Hexamethylenediamine (HMD, Aldrich), Isopropanol (VWR), 2-Iminothiolane hydrochloride (2-IT, Sigma), DL-Dithiothreitol (DTT, Sigma), Ethylenediaminetetraacetic acid anhydrous (EDTA, Sigma), 5,5-Dithiobis(2-nitrobenzoic acid (DTNB, Sigma),

Results and discussion

Due to the unique material properties of CS, the rational of the present work is the immobilization of CS on electrospun PCL NFMs surface to enhance the biological performance for culturing human chondrocytes. For that, we firstly modified the surface of PCL nanofibers by aminolysis. Although the aminolysis reaction was well established for PCL membranes [6], on electrospun PCL NFMs the reaction is not straightforward, requiring more extreme reaction conditions. Mattanavee et al. [19] performed

Conclusions

The immobilization of CS at the surface of electrospun PCL NFMs was successfully achieved with prior surface functionalization by exposure to UV/O3 and aminolyse. Physicochemical characterization of CS-immobilized PCL NFMs showed lower roughness and greater hydrophilicity than the samples without CS.

In vitro biological assays revealed that CS-immobilized PCL NFMs represents an efficient substrate for the maintenance of human articular chondrocytes phenotype, namely its typical round shape and

Acknowledgments

Juliana F. Piai would like to acknowledge CAPES-Brazil and CNPq-Brazil for the financial support (Proc. No. 201733/2009-9). Marta Alves da Silva and Albino Martins would like to acknowledge the Portuguese Foundation for Science and Technology (FCT), FCT/MCTES (POCH − Programa Operacional Capital Humano, comparticipado pelo Fundo Social Europeu e por fundos nacionais do MCTES) for their post-doctoral grants (SFRH/BPD/73322/2010 and SFRH/BPD/70669/2010). Authors would like to acknowledge the

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