Porous chitosan-gelatin scaffold containing plasmid DNA encoding transforming growth factor-β1 for chondrocytes proliferation

Abstract

Cartilage defects as a result of disease or injury have a very limited ability to heal spontaneously. Recently, tissue engineering and local therapeutic gene delivery systems have been paid much attention in the cartilage natural healing process. Gene-activated matrix (GAM) blends these two strategies, serving as local bioreactor with therapeutic agents expression and also providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In the current study, we used chitosan-gelatin complex as biomaterials to fabricate three-dimensional scaffolds and plasmid DNA were entrapped in the scaffolds encoding transforming growth factor-β1 (TGF-β1), which has been proposed as a promoter of cartilage regeneration for its effect on the synthesis of matrix molecules and cell proliferation. The plasmid DNA incorporated in the scaffolds showed a burst release in the first week and a sustained release for the other 2 weeks. The gene transfectd into chondrocytes expresses TGF-β1 protein stably in 3 weeks. The histological and immunohistochemical results confirmed that the primary chondrocytes cultured into the chitosan-gelatin scaffold maintained round and owned characters of high secretion of specific ECM. From this study, it can be concluded that this gene-activated chitosan-gelatins matrix has a potential in the application of cartilage defects regeneration.

Introduction

Articular cartilage has a simple architecture composed of a unique type of cell, the articular chondrocyte which disperses in an abundant extracellular matrix (ECM) mainly composed of proteoglycan aggrecan and type II collagen [1], [2]. Cartilage lesions resuling from acute or chronic injuries are one of the major factors leading to joint disease, disability, and eventually osteoarthritis. It is well known that adult articular cartilage has limited self-repair ability because of non-vascular supply. The healing of damaged cartilage still presents a clinical problem and many researchers are puzzled by reconstruction of hyaline cartilage. A few techniques available to repair cartilage defects have been used clinically, such as subchondral bone marrow stimulation [3], mosaicplasty [4], autologous chondrocyte implantation [5], osteochondral allograft [6] and artificial joint replacement techniques [7]. However, most of them have limitations with loss of more tissue or increasing injury. Recent efforts focused on the tissue engineering of articular cartilage applied a new approach for cartilage repair [8], [9].

Tissue engineering typically involves the fabrication of biological constructs that will restore, maintain and improve tissue function [10]. As we know that these regeneration processes are affected significantly by the local conditions such as the seeding cell resources, suitable scaffolds for cell adhesion and proliferation, and the nutrition supply. Because of non-blood supply in the cartilage defects, the supplies of nutritional substances are very limited. So the application of some growth factors will improve the local environment and enhance ex vivo synthesis of articular cartilage. Transforming growth factor beta1 (TGF-β1) is an important regulator of chondrocytes proliferation and differentiation and can increase the synthesis of specific ECMs [11], [12]. There are two approaches to introduce growth factors. One is to deliver recombinant proteins directly and the other is to use gene therapy technique. Gene therapy technique has more advantages in the local expression than continuously injecting recombinant growth factors since DNA is more stable and flexible than proteins and therefore likely to be compatible with established sustained delivery systems. To achieve a more sustained expression of such growth factors, plasmid DNA should be transfected to the seeding cells. Now non-viral transfection approaches including polymers and liposomes can offer high safety and enough transfection efficiency [13]. The gene-activated matrices (GAM) are a platform for gene delivery and as bioreactors for seeding cells to secrete plasmid-encoded proteins that enhance cartilage natural healing process. The encapsulated DNA content can be controlled by different ratios with polymers and the conditions in the incorporation process [14], [15]. This system combines tissue engineering with gene therapy for local sustained release of therapeutic gene which can be delivered to a particular compartment such as knee or ankle joint and minimizes side effects, and it also protects plasmid DNA from rapid enzymatic degradation, phagocytosis by synovial cells or macrophages in joint liquids.

Materials used for the slow release of plasmid DNA include poly (lactide-co-glycolide) (PLGA), alginate hydrogels, gels of polymethacrylic acid and polyethylene glycol, poly (ethylene-co-vinyl acetate), gelatin and collagen [16], [17], [18], [19], [20]. Chitosan is a biocopolymer comprising glucoseamine and N-acetylglucosamine, and gelatin (Gel) is a partially denatured derivative of collagen, which has low immunogencity, low cost and can be degraded entirely in vivo [21]. Chitosan and gelatin are chosen as GAM for cartilage regeneration in this study for the following reasons: (1) They have advantages to synthesize cartilage in vivo since their degradation products are non-toxic and similar to cartilage ECMs composition. Recent studies have demonstrated that these degraded products are involved in the synthesis of articular components, such as chondroitin sulfate, dermatan sulfate, hyaluronic acid, keratin sulfate and type II collagen. (2) They are all natural cationic matrices that can encapsulate more plasmid DNA. (3) Three-dimensional scaffolds can be fabricated with appropriate porousness and mechanical property for cartilage reconstruction with chitosan-gelatin complex. For example, chitosan increases the rate of gel formation and the strength of the resulting gels and gelatin changes the brittleness of chitosan reversely [22], [23], [24].

In the present study, we designed GAMs consisted of plasmid DNA and biodegradable materials as scaffolds for cartilage regeneration. The objective of this study is to investigate the primary chondrocytes cultured on the chitosan-gelatin three-dimensional scaffolds containing plasmid DNA encoding TGF-β1 by studying cells proliferation, adherence and the proteins expression. The plasmid DNA in the scaffolds can be partly protected from degradation by human serum and transfected to the primary chondrocytes. TGF-β1 protein can be expressed at a relatively high level for nearly 3 weeks. The chondrocytes cultured into scaffolds achieve proliferation and secret specific extracelluar matrices. The round cell shape is maintained all the time and the new cartilage-like tissue can be synthesized in the chitosan-gelatin gene-activated scaffolds.