Controlling the differentiation of human mesenchymal stem cells (hMSC) and providing tissue functions in engineered constructs before implantation are major challenges. Beside the additives in culture media, the artificial niches inside a scaffold can serve this purpose. To prepare niches favoring the osteoblastic differentiation of hMSCs, components of the extracellular matrix of bone were immobilized on fabrics of poly(3-hydroxybutyrate). Aqueous gels of fibrillar bovine collagen I, with or without addition of chondroitin sulphate (CS), were immobilized on the textile scaffold, sub-structured in a freeze-drying process, and cross-linked. hMSCs of four donors were isolated from bone marrow. After expansion, the cells were seeded dynamically onto the scaffolds. From thereon, the culture was transferred into perfused vessels and partly submitted to dexamethasone to promote osteogenic differentiation. During their 4 weeks of culture, the cells' distribution and morphology throughout the scaffolds were characterized by laser scanning microscopy (LSM) and scanning electron microscopy (SEM). Photospectrometrically the cells' viability (MTT) and alkaline phosphatase (ALP) production were assessed. The transcription of osteoblast-specific markers was elucidated with polymerase chain reaction (PCR) tests. Cells on CS-containing scaffolds in the presence of dexamethasone showed the highest ALP production. PCR monitored an increase of osteoblastic markers. All scaffolds showed higher calcium deposition than cell-free controls. These results lead to the conclusion that a niche containing CS renders the differentiation of hMSCs toward osteoblastic cells more specific.