Characterization of a Genetically Engineered Elastin-like Polypeptide for Cartilaginous Tissue Repair Helawe Betre, Lori A. Setton, Dan E. Meyer, and Ashutosh Chilkoti* Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708 Received January 8, 2002; Revised Manuscript Received May 9, 2002 Elastin-like polypeptides (ELPs) are artificial polypeptides with unique properties that make them attractive as a biomaterial for tissue-engineered cartilage repair. ELPs are composed of a pentapeptide repeat, Val- Pro-Gly-Xaa-Gly (Xaa is any amino acid except Pro), that undergo an inverse temperature phase transition. They are soluble in aqueous solution below their transition temperature (T t ) but aggregate when the solution temperature is raised above their T t . This study investigates the rheological behavior of an un-cross-linked ELP, below and above its T t , and also examines the ability of ELP to promote chondrogenesis in Vitro.A thermally responsive ELP with a T t of 35 °C was synthesized using recombinant DNA techniques. The complex shear modulus of the ELP increased by 3 orders of magnitude as it underwent its inverse temperature phase transition, forming a coacervate, or gel-like, ELP phase. Values for the complex shear moduli of the un-cross-linked ELP coacervate are comparable to those reported previously for collagen, hyaluronan, and cross-linked synthetic hydrogels. Cell culture studies show that chondrocytes cultured in ELP coacervate maintain a rounded morphology and their chondrocytic phenotype, characterized by the synthesis of a significant amount of extracellular matrix composed of sulfated glycosaminoglycans and collagen. These results suggest that ELPs demonstrate great potential for use as in situ forming scaffolds for cartilaginous tissue repair. Introduction Cartilaginous tissues, such as articular cartilage, the meniscus, and intervertebral disc, contribute to mechanical load support, load distribution, and flexibility in joints of the body. Cartilaginous tissues consist of an extracellular matrix composed largely of water, fibrillar and nonfibrillar collagens, and negatively charged glycosaminoglycans. 1 In addition, cartilage is avascular with a very low cell density that contributes to its virtual inability for self-repair. Con- sequently, tissue-engineered strategies based on exogenous scaffolds and cellular supplementation have been widely studied for cartilage repair. Natural polymers proposed for cartilage repair with or without cellular supplementation include alginate, fibrin glue, chitosan, hyaluronan, and type I and II collagen gels. 2-8 In addition, synthetic biomaterials, such as poly(glycolic acid), poly(lactide), poly(caprolactone), and various copolymers, have been widely studied. 9-13 Most of these polymers are formed ex ViVo and implanted into a defect of fixed size and shape, generally with suture fixation. More recently, Stile and co-workers, using cross-linked poly- (N-isopropyl acrylamide-co-acrylic acid) and Chenite and co- workers, using chitosan, have developed injectable hydrogels that may enable irregular cartilage defects to be filled without the need for precise surgical fixation. 14,15 Some of these biomaterials have been successful in promoting cellular infiltration and proliferation, and differentiation of either primary cartilage cells or chondrogenic progenitor cells, as assessed by histological appearance, biochemistry, and immunohistochemistry. An important goal of successful cartilage repair is restora- tion of the mechanical properties and, hence, mechanical function of the cartilaginous material. In particular, restoring mechanical function at the time of cartilage repair may be highly desirable for ensuring maintenance of tissue and joint function as well as the mechanical environment that governs cell metabolism and matrix homeostasis. 16 Thus, there is great interest in new technologies to manipulate the mechanical properties of a biomaterial during synthesis and implantation in order to obtain a functional scaffold that will support load and permit integration with native tissue. Elastin-like polypeptides (ELPs) are artificial polypeptides with unique properties that make them attractive as an injectable material for tissue-engineered cartilage repair. ELPs consist of oligomeric repeats of the pentapeptide sequence Val-Pro-Gly-Xaa-Gly, where Xaa is any amino acid except proline. This is a naturally occurring sequence in the protein elastin contained in muscle, ligaments, cartilage, and numerous other soft tissues. 17,18 Below a specific temperature, termed the inverse phase transition temperature (T t ), ELPs are soluble in aqueous solution. When raised above their T t , ELPs precipitate out of solution and form a gelatinous aggregate termed a coacervate. 19,20 Thus, the temperature- induced coacervation of ELPs may provide a simple method to create injectable materials for cartilage repair, with features that make them uniquely attractive as a functional tissue engineering scaffold: (1) below their phase transition tem- * To whom correspondence may be addressed: Department of Biomedi- cal Engineering, Box 90281, Duke University, Durham, NC 27708; phone, (919) 660-5131; fax, (919) 660-5362; e-mail, chilkoti@duke.edu. 910 Biomacromolecules 2002, 3, 910-916 10.1021/bm0255037 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/04/2002