Long-term intermittent shear deformation improves the quality of cartilaginous tissue formed in vitro Stephen D. Waldman a , Caroline G. Spiteri a , Marc D. Grynpas a,b , Robert M. Pilliar b , Rita A. Kandel a,b, * a Department of Pathology and Laboratory Medicine and Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ont., Canada M5G 1X5 b Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ont., Canada M5S 3G9 Abstract The formation of cartilaginous tissue in vitro is a promising alternative to repair damaged articular cartilage. However, recent attemptstotissue-engineerarticularcartilagethathassimilarpropertiestothenativetissuehaveproventobedifficult.Theinvitro- formed cartilaginous tissue typically has a similar proteoglycan content to native cartilage, but has a reduced collagen content and only a fraction of the mechanical properties. In this study, we investigated whether the intermittent application of cyclic shearing forces during tissue formation would improve the tissue quality. Chondrocyte cultures were stimulated at a 2% shear strain am- plitude at a frequency of 1 Hz for 400 cycles every 2nd day. At one week, both collagen and proteoglycan synthesis increased (23  6% and 20  6%, respectively) over the unstimulated, static controls. At four weeks, an increased amount of tissue formed (stimulated:1:85  0:08, unstimulated:1:58  0:07mgdrywt.).Thistissuecontainedapproximately40%morecollagen(stimulated: 511  23, unstimulated: 367  24 lg/construct) and 35% more proteoglycans (stimulated: 376  21, unstimulated: 279  26 lg/con- struct). Tissues that formed in the presence of shearing forces also displayed a 3-fold increase in compressive load-bearing capacity (stimulated:16  5, unstimulated:5  1 kPa max. equilibrium stress) and a 6-fold increase in stiffness (stimulated: 112  36, unsti- mulated:20  6 kPa max. equilibrium modulus) compared to the static controls. These results demonstrate that intermittent ap- plication of dynamic shearing forces over a four-week period improves the quality of cartilaginous tissue formed in vitro. Interestingly, low amplitudes of shear stimulation for short periods of time (6 min of stimulation applied every 2nd day) produced these changes. Ó 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. Keywords: Tissue-engineering; Articular cartilage; Calcium polyphosphate substrates; Chondrocytes; Shear; Mechanical stimulation Introduction Articularcartilageisahydratedconnectivetissuethat covers the articulating surfaces of synovial joints. Car- tilage has a limited capacity for self-repair [18], and the few surgical techniques available to repair damaged cartilage (e.g. mosaicplasty and autologous chondro- cyte implantation) have not proven entirely successful [25,32].Recenteffortsatcartilagerepairhavefocusedon tissue-engineering methods to create cartilaginous tissue in vitro suitable for joint resurfacing. One of these ap- proachesinvolvesculturingisolatedchondrocytesonthe surface of biodegradable substrates [41,42] or within synthetic [9,10,13,31,39,40] or biological scaffolds [6,7]. When grown in culture, the chondrocytes synthesize tissuethatcontainsextracellularmatrixmacromolecules similar to those present in articular cartilage [6,7,40, 41]. However, when compared to native cartilage, the in vitro-formed tissue typically contains less extracellu- lar matrix [1,6,7,31,40,41], which is deficient in collagen [1,31,40,41] and displays inferior mechanical properties [6,7,10,20,41]. The mechanical environment of the tissue regulates, in part, cartilage development and maintenance in vivo [17], leading to investigations on the effect of compres- sive forces on extracellular matrix synthesis in carti- lage explants [2,22,33,37,43] and scaffolds seeded with * Corresponding author. Department of Pathology and Laboratory Medicine and Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ont., Canada M5G 1X5. Tel.: +1-416-586-8516; fax: +1-416-586-8628. E-mail address: rkandel@mtsinai.on.ca (R.A. Kandel). 0736-0266/03/$ - see front matter Ó 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0736-0266(03)00009-3 www.elsevier.com/locate/orthres Journal of Orthopaedic Research 21 (2003) 590–596