Chitosan modified poly(l-lactide) microspheres as cell microcarriers for cartilage tissue engineering

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Abstract

The surfaces of poly(l-lactide) (PLLA) microspheres were modified by chitosan via a method of hydrolysis and grafting-coating to improve their compatibility to chondrocytes. The PLLA microspheres with a diameter of 74–150 μm were fabricated by an oil/water emulsion solvent evaporation method, followed by hydrolysis in alkaline solution to produce a larger number of carboxyl groups. Using water-soluble carbodiimide as a coupling reagent, chitosan was covalently grafted onto the microspheres. Due to the physical entanglement and insolubility at neutral pH, unbonded chitosan molecules were stably remained to yield a large amount of coated chitosan. Biological performance of the control PLLA and the chitosan-coated PLLA microspheres were assessed by in vitro culture of rabbit auricular chondrocytes. After 24 h and 7 d culture, the chitosan-coated PLLA microspheres, especially the ones with larger chitosan amount, exhibited stronger ability to promote cell attachment and proliferation, and maintain the secretion function of the chondrocytes. Therefore, the chitosan-coated PLLA microspheres can be potentially used as the injectable cell microcarriers for chondrogenesis in cartilage tissue engineering.

Introduction

Natural articular cartilage itself lacks a blood supply to support spontaneous self-repair. Tissue engineering and regenerative medicine have demonstrated their great success in regeneration of cartilage with normal function and bioactivity. In this process, a construct of chondrocytes/three-dimensional (3D) scaffold is often implanted into a defect site [1], [2], [3]. Therefore, the scaffolds play an important role in cartilage regeneration. There are various technologies for fabricating 3D porous scaffolds [4], including thermally induced phase separation (TIPS), freeze-drying, particle leaching, and 3D printing. More recently, injectable scaffolds have gained much attention due to their advantages of maintaining cell differentiated phenotype and minimal incision during the transplantation [5], [6], [7]. Biodegradable microspheres are a type of injectable materials, which can be used as cell microcarriers [5], [8], [9], [10], delivery system for drugs [11] and growth factors [12] as well as further assembly into porous scaffolds to form thick construct for cell infiltration and clinical applications [13]. The microspheres could serve as the cell microcarriers in tissue regeneration because of their unique properties for scaling up 3D cell culture and large surfaces for promoting cell expansion and maintaining cell phenotype [5], [8], [9], [10]. More importantly, microcarriers carrying cells can be directly injected into tissue defect site without undergoing an open surgery process.

Various types of microcarriers have been developed so far [8], of which synthetic polyesters including polylactide (PLA), polyglycolide (PGA) and poly(lactide-co-glycolide) (PLGA) are particularly attractive due to their good mechanical properties, processibility, biocompatibility and biodegradability [9], [10]. Acting as the cell carriers, however, surface modification of the polyesters such as poly(l-lactide) (PLLA) is still required to improve their bioresponsivity for cells. One of the appealing and effective strategies is to enrich their surfaces with bioactive components such as fibronectin (Fn) and RGD (Arg-Gly-Asp) peptide sequence [14], forming a biomimetic interface.

Chitosan is another frequently applied biomaterial. It is composed of glucosamine and N-acetylglucosamine, which is structurally similar to glycosaminoglycan (GAG) produced by chondrocytes and other analogs such as chondroitin 4-sulphate, chondroitin 6-sulfate and keratin sulfate [14], [15], [16]. GAG is one of the principle components of the normal cartilage-specific extracellular matrix (ECM) and can stimulate chondrogenesis [17]. Therefore, similar bioactivity of chitosan to GAG can be expected. Previous results show that the chitosan-based constructs including 3D porous scaffolds [18], hydrogels [19] and microcarriers [20] are beneficial of wound healing. We also found that a chitosan-g-lactose/heparin film could accelerate chondrocyte growth with preserved phenotype [21]. Moreover, the large number of amino groups on the chitosan molecules makes further modification possible by methods of covalent bonding [22], layer-by-layer assembly [23], plasma treatment [15] and grafting-coating [9], [24].

Herein PLLA microspheres are modified to obtain cell carriers, which can be used as cell delivery vehicle and an injectable scaffold. Surface hydrolysis of the microspheres produces abundant carboxyl groups, by which chitosan is immobilized by a grafting-coating technique (Scheme 1). In vitro culture of rabbit auricular chondrocytes is finally conducted to assess their biological performance.

Section snippets

Materials

Poly(l-lactide) (PLLA; Mn = 200 kDa and Mw = 400 kDa) was purchased from China Textile Academy. Chitosan (deacetylation degree 85%, Mn = 6.2 × 105) was obtained from Haidebei Bioengineering Co. (Qingdao, China). Its density was measured as 1.42 g/cm3 by a bottle method. Poly(vinyl alcohol) 124 (PVA 124, average Mw = 85–124 kDa and 98–99% hydrolyzed) was supplied by Shanghai Medicine and Chemical Company, China. p-Dimethylaminobenzaldehyde was supplied by Shanghai San’aisi Chemical Company, China.

Surface hydrolysis of PLLA microspheres

To overcome the disadvantage of poor cell–material interaction, the PLLA microspheres were stably coated with a chitosan layer by a grafting-coating technique. The surface hydrolysis breaks some of the ester groups, yielding carboxyl (–COOH) and hydroxyl (–OH) groups on the PLLA chain termini, by which immobilization of bioactive components such as proteins, enzymes, growth factors, polysaccharides and peptides becomes possible (Scheme 1). Fig. 1 shows that amount of the carboxyl groups (–COOH)

Conclusions

Chitosan-coated PLLA microspheres have been successfully prepared by a method of surface hydrolysis and grafting-coating. The hydrolysis produces more carboxyl groups and needle-like cracks on the microsphere surfaces. With still longer reaction time such as 50 min, severe erosion and weight loss takes place. The immobilized chitosan is 8.7 ± 2.1 μg/mg and 14.2 ± 2.4 μg/mg microspheres on the PLLA-chit2 microspheres and the PLLA-chit10 microspheres, respectively, which correspond to a surface chitosan

Acknowledgements

This study is financially supported by the Major State Basic Research Program of China (2005CB623902), the National High-tech Research and Development Program (2006AA03Z442), the Science and Technology Program of Zhejiang Province (2006C13022), and the National Science Fund for Distinguished Young Scholars of China (No. 50425311).

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