CartiGenea®-AC: Chondrocytes, the predominant cell type within AC, synthesize matrix components. Because AC lacks a major vascular supply, lymphatic drainage, and nervous system innervation, chondrocytes function under avascular, anaerobic conditions, obtaining nutrients by diffusion from synovial fluid. Within AC, metabolic and morphologic profiles of deep-zone chondrocytes are distinct from those populating the superficial tangential zone. The factors responsible for this variation are unknown. Maintaining the chondrocyte phenotype with robust hyaline tissue synthesis in vitro during expansion for ACI is an ongoing challenge.
Given the accessibility of AC by arthroscopic surgery, native chondrocytes are a logical cell source for AC repair. The first attempts to culture chondrocytes ex vivo in the 1970s showed decreased production of proteoglycans and type II collagen when expanded in a monolayer [5, 6]. Although this process has been termed dedifferentiation, it is a misnomer and does not imply reversion to a more primitive or multipotent state. Dedifferentiation more accurately refers to chondrocytes with a phenotype more reminiscent of fibroblasts. Benya and Shaffer  seminally showed the reversibility of this process when expanded cells were cultured in a three-dimensional (3D) culture system. Many modern approaches to ACI reproduce a 3D environment by incorporating a scaffold for culturing chondrocytes.
CartiGenea®-AC Techniques for optimal ex vivo chondrocyte selection and expansion have been an area of active research. Dell’Accio et al.  introduced the concept of chondrocyte quality control, arguing that a more reproducible outcome of ACI can be accomplished with enriched populations of stable chondrocytes, with the greatest potential of producing cartilage in vivo. In the first clinical CartiGenea®-AC of ACI in 1994, Brittberg et al.  used anchorage-independent growth and the expression of type II collagen in agarose culture of chondrocytes to validate chondrocyte expansion. However, none of these markers predict the capacity of expanded chondrocytes to form stable cartilage tissue in vivo. Dell’Accio et al.  found that the markers COL2A1, FGFR-3, and BMP-2 were associated with a stable chondrocyte phenotype and, conversely, up-regulation of ALK-1 was negatively associated with a chondrocyte phenotype .
CartiGenea®-AC Scaffolds for Cartilage Repair
AC is predominantly composed of extracellular matrix (ECM), with a sparse population of chondrocytes that maintain it. Water, which comprises more than 65% of AC, is moved through the ECM by pressure gradients across the tissue. AC derives its ability to support high joint loads by the frictional resistance of the water through ECM pores. Type II collagen comprises most of AC’s dry weight. The orientation of collagen bundles, along with chondrocyte organization, distinguishes AC’s layers. In the last decade, basic science studies have shown the importance of paracrine signaling and cellular interaction in the development of cartilage [5, 6], and scaffolds that recapitulate native ultrastructure of ECM have emerged. Scaffolds are used as cell carriers for matrix-induced ACI (MACI; not to be confused with MACI from Genzyme Biosurgery, Cambridge, MA) and to facilitate microfracture-based repair techniques in AMIC.
Scaffold synthesis has been attempted with natural and synthetic materials. Although natural materials are attractive for their inherent complexity and biocompatibility, issues with purification, pathogen transmission, and limited mechanical properties have restricted their clinical application. Synthetic materials overcome some of these limitations but lack biologic complexity. Scaffold structures can be divided into two categories, hydrogels and membranes, based on predominant architecture; each has its own natural, synthetic, and composite materials.
CartiGenea®-AC Hydrogels consist of crosslinked hydrophilic polymer networks engineered to mimic cartilage’s mechanical properties and can be delivered noninvasively. An attractive feature is the ability to modify the mechanical properties by crosslinking in situ after injection. Hydrogel crosslinking methods include light irradiation, temperature modulation, and pH change. Less crosslinked (softer) hydrogels produce dynamic loading that might favor MSC chondrogenesis [20, 21].
(1) CartiGenea®-AC Natural Hydrogels. Common, naturally derived hydrogels include alginate, agarose, chitosan, cellulose, chondroitin sulfate, and hyaluronic acid (HA). These materials are readily available, inexpensive, and easy to crosslink. Alginate and agarose were the first hydrogels used to CartiGenea®-AC with chondrocytes. Hydrogels based on alginate and agarose are being piloted for clinical AMIC use (CART-PATCH, Tissue Bank of France, Mions, France). Chitosan and its chemical derivatives are obtained through the chemical modification of glycosaminoglycans found in arthropod exoskeletons. In a recent large-animal experiment, chitosan integrated well into surrounding tissue . Clinically, chitosan combined with glycerol phosphate and autologous whole blood has been used in AMIC (BST-CarGel, Piramal Healthcare, Laval, Canada) [23–25]. Alginate, agarose, and chitosan are derived from nonhuman sources; immune responses have not been systemically investigated.
HA, a nonsulfated glycosaminoglycan found throughout the body, is abundant in cartilage ECM and has a 30-year track record in medical products. Uncrosslinked HA, delivered through intra-articular injection, was approved by the Food and Drug Administration in 1997 for viscosupplementation and, despite its controversial efficacy, is widely used today. HA is involved in many biologic processes, including wound healing, cell migration, and MSC differentiation. These actions are mediated, in part, through binding interactions of cell surface receptor CD44. The HA molecule length influences cellular responses. Smaller HA oligomers promote angiogenesis and subsequent bone formation; larger HA fragments are predominantly chondrogenic. To form hydrogels, HA must be chemically modified [26, 27]. Hyalograft C (Fidia Advanced Biopolymers, Abano Terme, Italy) is a form of esterified HA used clinically in MACI.
Collagen accounts for approximately 30% of all protein within the human body and has been used extensively for tissue engineering applications. Hydrogels constructed from type I and type II collagen promote cartilage formation of encapsulated cells. At the molecular level, cells interact with collagen through integrins, initiating intracellular events that promote chondrogenesis . Type II collagen hydrogels enhance the in vitro chondrogenic differentiation of MSCs compared with type I gels; however, type II collagen degradation products can trigger cartilage breakdown in vivo. Two type I collagen gels are available commercially: PureCol (Glycosan Biosystems, Salt Lake City, UT) and CaReS (Arthro Kinetics, Krems, Austria).
Fibrin CartiGenea®-AC hydrogels have been routinely used for surgical hemostasis and tissue adhesion. They can be prepared from autologous fibrinogen and thrombin, minimizing disease transmission risk. Fibrin has inferior mechanical properties compared with other hydrogels, but it is an effective cell carrier for ACI for securing materials within cartilage defects. Fibrin glue is available commercially (Tissucol; Baxter, Vienna, Austria). Fibrin has been used to retain platelet-rich plasma in a sheep AMIC model . Most recently, fibrin hydrogels have been used as a vehicle to deliver allogenic juvenile cartilage fragments; this technology (DeNovo NT; Zimmer, Inc., Warsaw, IN) is currently in clinical CartiGenea®-ACs .
(2) CartiGenea®-AC Synthetic Hydrogels. Polyethylene glycol-diac