General introduction
Normal articular cartilage is avascular, anuronal tissue. And it is complex and consists of chondrocytes and cartilage specific extracellular matrix (ECM) which is mainly composed by collagens and proteoglycans (Fig. 1). Articular cartilage in the knee plays a key role to absorb and distribute various mechanical loads in the joint.
Unfortunately, once damaged adult human articular cartilage show a poor capacity for repair and regeneration. Explanation for this include that the limited potential for chondrocyte proliferation, the capacity chondrocytes to become catabolic in response to pathological mediators, and the avascular nature of the tissue. Until now, various surgical procedures such as debridgement, subchondral microfracture and drilling, auto or allogenous osteochondral transplantation and autologous chondrocytes implantation have been developed to help regeneration of damaged cartilage. In fact, so far no perfect methods and results have been found to substitute the shapely articular cartilage normally present in the joint.
Surface of cartilage Chondrocyte Collagen
Proteoglycan Proteoglycan Chodrocyte
Fig. 1. Articular cartilage. (A) surface of articular cartilage; (B) Safranin-O staining;
(C) Molecular level of articular cartilage.
Recently, the tissue engineering technology has been a strong candidate as regeneration for damaged articular cartilage.
The tissue engineering technique first introduced in the late 1980s. It is a multidisciplinary research area that incorporates both biological and engineering principle for the purpose of generating new, living tissues to replace the diseased/damaged tissue and restore tissue/organ function. So far, tissue engineering technique was widely used in various filed of regenerative medicine. Among them, for successful articular cartilage tissue regeneration four tools are necessary (Fig. 2): i) tissue specific cells including stem cells (embryonic stem cells (ESC), mesenchymal stem cells (MSC), umbilical cord stem cells (UCSC)), chondrocytes, perichondrials cells, periosteum; ii) Biocompatible carrier scaffolds by which seeded cells are supported and can develop. It is including natural materials such as small intestinal submucosa (SIS), De-mineralized bone matrix (DBM), human amniotic membrane (HAM), fibrin, collagen, and synthetic materials such as polyglycolic acid (PGA), polylactic acid (PLA) and polylactic-glycolic acid (PLGA); iii) Signaling molecules including growth factors (tissue growth forming factors (TGFs), insulin-like growth factors (IGFs), bone morphogenetic proteins (BMPs), fibroblasts growth factors (FGFs)), cytokines, and non-proteinaceous chemical compounds; iv) Bioreactors including hydrodynamic, dynamic compressive loading, hydrostatic pressure et al.
Fig. 2. Component of tissue engineering.
Unfortunately, the perfect conditions have not been found for formation of high quality articular cartilage until now. Anyway, we consider that the two points as cells and scaffolds is most importance in the cartilage tissue engineering. Chondrocytes is already differentiated cells and widely used in clinic for cartilage regeneration, so that we think it is the best choice in current stage. In other hand, an ideal carrier material either in vitro or in vivo should provide several characteristics including mechanical stability, biodegradability, biocompatibility, guarantee of uniform cell distribution and avoid influence on seeded cells phenotype. In other words, it can provide not only structural guidance for cell growth and tissue morphogenesis, but can also play a functional role such as enhancing the cell attachment and the metabolism. We consider that this condition might be provided by the significance of extracellular matrix (ECM)
natural scaffolds. Because, the ECM scaffold as a nature’s natural scaffold, and it has high tissue compatibility and a potential to retain cytokines, growth factors, and other functional proteins. Therefore, the many ECM scaffolds such as SIS, HAM and DBM has been already noticed in the tissue-engineering field recently.
In present study, we made a novel cell-derived extracellular matrix (ECM) scaffold using porcine chondrocyte and freeze-drying technique, and evaluated the feasibility of the scaffold on cartilage tissue engineering in vitro and in vivo as nude mouse and cartilage degenerates and often advances to osteoarthritis (OA) without appropriate cares, because it has very poor self-healing capability. Many researchers have investigated a tissue engineering approach using cultured cells and/or various scaffolds with the ultimate aim of promoting the regeneration of injured cartilage. The scaffolds used include both synthetic and natural polymers such as polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), fibrin gel, collagen, alginate, chitosan, hyaluronic acid (HA) and collagen-glycosaminoglycan (GAG) composite, etc (Nehrer et al 1997; Rotter et al 1998; Honda et al 2000; Lee et al 2000; Griogolo et al
2001; Liu et al 2004; Park et al 2005; Li and Zhang 2005). Although some successful results were reported using these scaffolds in vivo and in vitro environments, no satisfactory scaffold is currently available that can regenerate high quality cartilage tissue.
The significance of ECM scaffolds has been recently noticed in the tissue-engineering field. As a nature’s natural scaffold, the ECM scaffold has high tissue compatibility and a potential to retain cytokines, growth factors, and other functional proteins. Therefore, it can provide not only structural guidance for cell growth and tissue morphogenesis, but can also play a functional role such as enhancing the cell attachment and the metabolism. For these reasons, some ECM scaffolds have been already used successfully in preclinical animal studies and clinical applications. For example, the small intestinal submucosa (SIS) was used successfully for urinary tract, dura mater, and vascular reconstructions (Prevel et al 1994;Cobb et al 1996; Kropp et al 1996), and the human amniotic membrane (HAM) was used successfully for corneal and cartilage regeneration (De Rotth 1940; Lee and Tseng 1997; Jin et al 2007). These ECM scaffolds were derived from tissues and used directly or with some modifications.
However, natural cartilage cannot be used as a scaffold on account of its peculiar dense structure and shape. This was the driving basis of our interest to make a cell-derived ECM scaffold using chondrocytes. It was hypothesized that a cell-derived ECM scaffold can provide a favorable 3-dimensional (3-D) environment to support the retention of chondrocytic phenotype, the synthesis of the ECM components, and the
formation of hyaline cartilage, and eventually to make a natural cartilage-like structure.
The cell-derived ECM scaffold was made using porcine chondrocytes and evaluated as a potential scaffold for cartilage tissue engineering in vitro. Its cellular compatibility and construction of a neocartilage-like structure were analyzed together with its morphological, physical and chemical properties.
Chapter II
Adult articular cartilage has a very limited healing potential. Tissue engineered cartilage has been a strong candidate as a replacement material for articular cartilage defects. Cartilage tissue engineering requires a multidisciplinary combination of medium supplements, growth factors, cell sources, and scaffolds, among which scaffolds play a pivotal role. Previously, various scaffolds have been utilized in the forms of gels, sponges, fibers, and microspheres (Griogolo et al 2001; Chen et al 2003;
Honda et al 2004; Kang et al 2005). Generally, scaffolds require a 3-dimensional (3D) structure, high porosity with an interconnection, balanced biodegradability, and biocompatibility. Although a perfect material for scaffolds was developed in the tissue engineering field, natural extracellular matrix (ECM) scaffolds has gained a prominent interest for long time. Because the ECM scaffolds could provide specific environments similar to natural tissues both structurally and functionally, the small intestine submucosa (SIS), urinary bladder (UBS), and human amniotic membrane (HAM) have been used for the reconstruction of vascular, bladder, tendon, and corneal tissues
(Badylak et al 1995; Kopp et al 1996; Lee and Tseng 1997; Piechota et al 1998;). In addition, a mixed scaffold of collagen and glycosaminoglycan (GAG) was also used recently (Lee et al 2000; O`Brien et al 2005; Zhong et al 2005).
In this report, we utilized a different strategy to make ECM scaffolds compared to the previously ones. Instead of using tissue itself as a scaffold, chondrocyte cells cultured in vitro was processed to form a scaffold that had the most similar structure to that of the
natural cartilage. In our previous study, we had introduced a well-fabricated cartilage from porcine articular chondrocytes without any scaffolds (Park et al 2006). We developed this into a highly porous, sponge type, cell-derived ECM scaffold with a freezing and drying technique (Jin et al 2006). We hypothesized that this cell-derived ECM scaffold could be able to provide an ideal 3D environment forming a hyaline cartilage for the cartilage tissue engineering. This study was designed to observe the process of cartilage formation after inoculation of chondrocytes on the scaffold and to evaluate its resemblance to the natural cartilage in terms of morphology, chemistry, and mechanical strength.
Chapter III
Articular cartilage injury is hard to repair due to the poor self-healing capacity of the cartilage, which easily causes degeneration of intact cartilages at the border resulting in osteoarthritis (OA) (Mankin 1982). Many surgical methods such as debridgement (Insall 1974; Baumgaertner et al 1990), subchondral microfracture and drilling
(Mitchell and Shepard 1976) have been reported to help regeneration of damaged cartilage. However, the regenerating tissues usually changed to the fibro-cartilage losing biomechanical properties of normal articular cartilage.
The osteochondral grafts technique suffered from the incongruence of the joint surface between the graft and the host cartilage, and the limitation in the source of donor tissues (Garrett 1994). In 1994, Britberg et al reported excellent results with autologous chondrocytes transplantation (ACT) technique (Brittberg et al 1994).
However, several questions still remain in this procedure overall such as the phenotypic changes in chondrocytes during in vitro culture, the possible leakage of the transplanted chondrocytes from the graft site, uneven distribution of chondrocytes and periosteal hypertrophy of them on the graft site, etc. These problems have led many researchers search for exogenous biocompatible materials to deliver chondrocytes to the lesion site for tissue engineering technique. Until now, a number of biocompatible materials have been used for cartilage repair including natural materials such as fibrin, collagen gels and sponges (Nehrer et al 1997; Lee et al 2000; Park et al 2005), and synthetic materials such as polyglycolic, polylactic acid and polylactic-glycolic acid (Rotter et al 1998;
Honda et al 2000; Liu et al 2004). Unfortunately, however, no biocompatible materials were reported showing satisfactory results of cartilage regeneration in vivo.
In our previous study, the feasibility of a novel cell-derived ECM scaffold in cartilage tissue engineering was demonstrated in vitro and in vivo in nude mouse (Jin et al 2006; 2007). This study investigated the potential of the ECM scaffold to induce
cartilage regeneration in rabbit defect model in particular depending on their maturity in vitro. The cartilage is resilient load-bearing material to absorb mechanical shock and spread the applied load onto bone in the joint. So, the properties of tissue engineered cartilage before implantation such as the chemical composition and mechanical strength could be very important in the result of cartilage regeneration in vivo. For example, immature engineered cartilage is likely to integrate itself well with the surrounding tissues, but could be easily broken down by the mechanical load. In contrast, mature cartilage might be more resistant to the mechanical load, thereby more excellent in regenerating the cartilage in vivo. Tissue-engineered cartilages with different maturity were prepared in vitro using the ECM scaffold and allogenous rabbit chondrocytes, and implanted into the cartilage defect in the rabbit trochlear groove to evaluate their regenerative capacity in vivo.