The Effect of Insulin-Loaded Chitosan Particle–Aggregated Scaffolds in Chondrogenic Differentiation Patrı ´cia B. Malafaya, Ph.D., 1,2 Joa ˜ o T. Oliveira, Ph.D., 1,2 and Rui L. Reis, Ph.D. 1,2 Osteochondral defect repair requires a tissue engineering approach that aims at mimicking the physiological properties and structure of two different tissues (cartilage and bone) using a scaffold–cell construct. One ideal approach would be to engineer in vitro a hybrid material using a single-cell source. For that purpose, the scaffold should be able to provide the adequate biochemical cues to promote the selective but simultaneous differenti- ation of both tissues. In this work, attention was paid primarily to the chondrogenic differentiation by focusing on the development of polymeric systems that provide biomolecules release to induce chondrogenic differen- tiation. For that, different formulations of insulin-loaded chitosan particle–aggregated scaffolds were developed as a potential model system for cartilage and osteochondral tissue engineering applications using insulin as a potent bioactive substance known to induce chondrogenic differentiation. The insulin encapsulation efficiency was shown to be high with values of 70.37  0.8%, 84.26  1.76%, and 87.23  1.58% for loadings of 0.05%, 0.5%, and 5%, respectively. The in vitro release profiles were assessed in physiological conditions mimicking the cell culture procedures and quantified by Micro-BCAÔ protein assay. Different release profiles were obtained that showed to be dependent on the initial insulin-loading percentage. Further, the effect on prechondrogenic ATDC5 cells was investigated for periods up to 4 weeks by studying the influence of these release systems on cell morphology, DNA and glycosaminoglycan content, histology, and gene expression of collagen types I and II, Sox-9, and aggrecan assessed by real-time polymerase chain reaction. When compared with control conditions (unloaded scaffolds cultured with the standard chondrogenic-inducing medium), insulin-loaded scaffolds up- regulated the Sox-9 and aggrecan expression after 4 weeks of culture. From the overall results, it is reasonable to conclude that the developed loaded scaffolds when seeded with ATDC5 can provide biochemical cues for chondrogenic differentiation. Among the tested formulations, the higher insulin-loaded system (5%) was the most effective in promoting chondrogenic differentiation. Introduction O steochondral defects affect both the articular carti- lage and the underlying subchondral bone in the joint area and may inflict pain and limited mobility, thereby compromising quality of life. The requirements for a suc- cessful regeneration of an osteochondral defect could po- tentially be met by using a tissue-engineered osteochondral (bone–cartilage) composite of predefined size and shape, generated in vitro using autologous cells. In this sense, vari- ous strategies have been reported that result from the use of one or more cell types cultured into single-component or more complex scaffolds in a broad spectrum of compositions and biomechanical properties as recently reviewed. 1,2 The most attractive approach seems to be the design of a suitable single scaffold that will provide differentiated and adequate conditions for guiding the growth of the two tissues, ful- filling their different biological and functional requirements. Ideally, the scaffold must be able to provide the different and appropriate differentiation cues to promote an adequate development of both cartilage and bone tissues, so that the final hybrid construct can be achieved with the same cell source. Several works 3–7 can be found reporting the use of dif- ferent biomolecules to promote the cell differentiation either as supplements of the culture medium or as loaded in the scaffolds. For instances, Martin et al. 7 have applied specific regulatory molecules to selectively differentiate bone marrow stromal cells into either cartilaginous or bone-like tissues in conjunction with three-dimensional porous polymeric struc- tures. The work shows that using appropriate chondrogenic (dexamethasone, insulin, and transforming growth factor-b1) or osteogenic (dexamethasone and b-glycerophosphate) me- dium supplements, the generated extracellular matrix (ECM) 1 3B’s Research Group, Department of Polymer Engineering, University of Minho, headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimara ˜ es, Portugal. 2 PT Government Associated Laboratory, Institute for Biotechnology and Bioengineering (IBB), Guimara ˜ es, Portugal. TISSUE ENGINEERING: Part A Volume 16, Number 2, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2008.0679 735