Journal of Cellular Biochemistry 83:121±128 (2001) Enhanced Cartilage Tissue Engineering by Sequential Exposure of Chondrocytes to FGF-2 During 2D Expansion and BMP-2 During 3D Cultivation Ivan Martin, 1,2 R. Suetterlin, 3 W. Baschong, 3 M. Heberer, 2 G. Vunjak-Novakovic, 1 and L. E. Freed 1 * 1 Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 2 Research Division, Department of Surgery, University of Basel, Switzerland 3 M.E. Mueller Institute, Biozentrum, University of Basel, Switzerland Abstract Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml ®broblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein-2 (BMP-2). Chondrocytes expanded without FGF- 2 exhibited high intensity immunostaining for smooth muscle a-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile ®broblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of ®broblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds. J. Cell. Biochem. 83: 121±128, 2001. ß 2001 Wiley-Liss, Inc. Key words: chondrocyte differentiation; growth factors; smooth muscle alpha actin; polymer scaffolds Chondrocytes in articular cartilage synthe- size collagen type II and large sulfated proteo- glycans, whereas the same cells cultured in monolayers (2D) dedifferentiate into ®broblas- tic cells and express collagen type I and small proteoglycans [von der Mark et al., 1977]. Chondrocyte dedifferentiation can be prevented or reversed by culturing cells in a 3D environ- ment supporting spherical cell morphology, such as agarose gels [Benya and Shaffer, 1982; Bonaventure et al., 1994] or biodegradable polyglycolic acid (PGA) scaffolds [Freed et al., 1994; Martin et al., 1999]. We recently showed that bovine calf articular chondrocytes expanded in 2D in the presence of FGF-2 fully redifferentiated and deposited col- lagen type II when cultured on 3D PGA scaf- folds, whereas the same cells expanded without FGF-2 remained elongated, developed thick F- actin structures, and induced shrinkage of the same scaffolds [Martin et al., 1999]. Canine chondrocytes expanded in 2D without FGF-2 also induced shrinkage of sponges made of collagen and GAG [Nehrer et al., 1997; Lee et al., 2000]. Chondrocyte-mediated extracellu- lar matrix contraction was recently proposed to be related to the expression of smooth muscle a- actin (SMA) [Lee et al., 2000], a contractile actin isoform typically expressed by specialized ®bro- blastic cells capable of applying tractional forces [Sappino et al., 1990]. Bone morphogenetic proteins (BMPs) speci®- cally regulate the early commitment of me- senchymal cells toward the chondrogenic and osteogenic lineages [Celeste et al., 1990]. In ß 2001 Wiley-Liss, Inc. DOI 10.1002/jcb.1203 Grant sponsor: National Aeronautics and Space Adminis- tration; Grant number: NCC8-174. *Correspondence to: L.E. Freed, M.I.T. Building E25, Room 330, 45 Carleton St., Cambridge, MA, 02139. E-mail: Lfreed@mit.edu Received 5 January 2001; Accepted 2 May 2001