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A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also theywere able to spread out from themicrospheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration. © 2011 American Chemical Society.