Introduction: The objective was to create multilayer cellular constructs using fetal or adult, decellularized articular cartilage in particulate form as microcarriers for expansion and fusion of mesenchymal stem cells (MSCs) to regenerate the stratified structure of articular cartilage. Methods:  Porous microparticles (CMPs) generated from decellularized fetal or adult bovine articular cartilage were used as microcarriers for expansion of human MSCs. The CMP expanded MSCs (CMP-MSCs) were used to generate injectable hydrogels or preformed multilayer constructs for articular cartilage regeneration. In the injectable approach, CMP-MSCs were suspended in alginate gel, crosslinked with calcium chloride, and incubated in chondrogenic medium to generate an injectable regenerative construct. In the preformed approach, fetal or adult CMP-MSCs were suspended in a culture medium, allowed to settle sequentially by the force of gravity, and fused by incubation in chondrogenic medium to generate multilayer cell sheets. The constructs were characterized with respect to compressive modulus, cellularity, and expression of chondrogenic markers. Results: Human MSCs expanded on fetal or adult CMPs in basal medium maintained the expression of mesenchymal markers. The injectable CMP-MSCs hydrogels had significantly higher expression of chondrogenic markers and compressive modulus after four weeks incubation in chondrogenic medium compared to MSCs directly encapsulated in alginate gel; preformed CMP-MSCs cell sheets had significantly higher compressive modulus and expression of chondrogenic markers compared to MSCs in the pellet culture. Conclusion: The preformed cell sheet approach is potentially useful for creating multilayer constructs by sequential gravitational settling of CMP-MSCs to mimic the stratified structure of articular cartilage.