Elsevier

Biomaterials

Volume 26, Issue 9, March 2005, Pages 987-998
Biomaterials

The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan

https://doi.org/10.1016/j.biomaterials.2004.04.015Get rights and content

Abstract

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, 1H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5–1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.

Introduction

Tissue-engineering procedures represent the actual trend in articular cartilage repair [1], [2], [3]. Accordingly, isolated autologous chondrocytes or mesenchymal stem cells are seeded in vitro in a 3D scaffold and induced to differentiate, to grow and to produce new extracellular matrix; the ensuing tissue grafts are suitable for in vivo implantation [4], [5]. Alternatively allogenic chondrocytes can be seeded in a suitable 3D matrix and immediately implanted into cartilage defects to rebuild, in vivo, a functional tissue, resembling the native cartilage and perfectly integrated with it [4], [5], [6], [7].

A basic requirement for tissue-engineering development is the availability of suitable 3D biodegradable matrices. Artificial or natural biomaterials have been extensively used [4], [5], in order to enhance biological interactions with cells and to improve cytocompatibility [8]. Fibrin, hyaluronan, collagen, calcium alginate gels or artificial polymers (like biodegradable polyesters of α-hydroxyacids) have shown to support tissue growth and repair. Beneficial effects of polysaccharides as protecting agents against oxygen derived free radicals, chondrolysis, prostaglandin synthesis and as cell-supporting matrices have been reported [9], [10], [11], [12]. However, the production of a sufficiently long-lasting hyaline cartilage of optimal mechanical strength has not yet been achieved. Therefore, many efforts have been directed towards the production of a biomaterial for cartilage engineering, having good similarity with the normal components of the natural matrix.

The attractive field of “biopolymer engineering” usually refers to the modification of a biopolymer chain introducing cell-specific ligands or extracellular signaling molecules, such as peptides and oligosaccharides. In particular, implication of oligosaccharides in cell signaling mechanisms and in recognition processes address them as key molecules in designing and developing a third-generation biomaterial [13], [14]. These materials are being designed to stimulate specific cellular response, to directly intervene in cell growth, differentiation, adhesion [15] and extracellular matrix production [16], [17] and therefore to improve cell embedding.

Among the wide number of polysaccharides available as potential candidates for biopolymer engineering, glycosaminoglycans (GAGs) belonging to the family of β(1–4)-linked linear polysaccharides, appear as a highly promising tool. In particular, chitosan, composed of 2-amino-2-deoxy-d-glucose (GlcNH2) residues with a variable number of randomly located N-acetyl-glucosamine groups (GlcNAc), revealed neither pathological inflammatory response in implantation models nor evidence of induced infection or presence of endotoxins and a low, if any, incidence of immunological reactions [18]. Recently chitosan and chitosan-based materials have been tested as biomaterials mimicking the cartilage matrix for a potential application in the repair of articular cartilage [19], [20].

The most interesting feature of chitosan as biomaterial is related to the presence of amino groups deriving from glucosamine units. The physico-chemical and biological properties of the polycation can be drastically modified exploiting the reactivity of glucosamine residues. Examples include acylation [21], [22], alkylation [23], [24] carboxymethylation [25] and quaternarization [26] of chitosan.

In this study we report on the biological properties of a chitosan sample modified by grafting of lactose via a reductive N-alkylation. Chitlac induced cell aggregation of a primary culture of pig chondrocytes and stimulated synthesis of chondro-specific GAGs and collagen. These results address chitlac as a new form of cell-delivery system for chondrocyte implantation in defective cartilage.

Section snippets

Materials

Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FCS), penicillin, streptomycin, trypsin/EDTA solutions, phosphate-buffered saline (PBS) and glutamine were purchased from Biochrom KG Seromed (Germany). Hyaluronidase, fluoresceine isothiocyanate (FITC) and Taq polymerase were from Sigma, (USA). Moloney murine leukemia virus reverse transcriptase (MLV-RT) was from Life technologies (USA). Collagenase type II was from Worthington Biochemical Corp. (USA). Chitosan and sodium

Synthesis and characterization of chitlac A and chitlac B

The two N-alkyl derivatives (chitlac A and B, Table 1) of purified chitosan, prepared as depicted in Scheme 1 were characterized by 1H-Nuclear Magnetic Resonance analysis, potentiometric titration and viscosity measurements.

Fig. 1 shows the 1H-NMR spectra of chitosan and chitlac A. From the ratio between the area of the peak assigned to H1 of GlcNH2 (4.49 ppm) and to H1 of GlcNAc (4.65 ppm) [29], [36] the residual chitosan acetylation degree (Fig. 1a) was found to be 11.3%.

The anomeric region of

Discussion

During the past decade exciting new strategies have emerged that have the potential to revolutionize the treatment of failure of tissue functions. The basic knowledge gained in the fields of cell and molecular biology, has provided a practical approach of bioregeneration focused on the delivery or in situ mobilization of capable cells to restore function tissues. This approach comprises the interactive triad of responsive cells, a supportive matrix and bioactive molecules promoting regeneration

Conclusions

A lactose derivative of a highly deacetylated chitosan, chitlac, showed very interesting biological effects towards a primary culture of pig chondrocytes. Beside its inhibitory effects on cell growth in short term cultures (up to 8–24 days) it was neither toxic for chondrocytes, nor inhibitor of the GAGs and collagen biosynthesis; to the contrary it stimulated the biosynthesis of chondro-specific aggrecan and type II collagen. A unique property appeared to be the induction of chondrocyte

Acknowledgements

This work was supported by grants of the University of Trieste and by the “Agenzia Spaziale Italiana (Research Contract I/R/371/02). We thank Dr. D. Pirulli (IRCCS, Burlo Garofalo, Trieste) for assistance in performing real time PCR.

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