This study aims to prepare biphasic osteochondral scaffolds based on seamless joining of sintered polymer and polymer/ceramic microspheres for co-culture of chondrocytes and bone marrow stem cells (BMSCs). up to 28 days. Scaffolds sterilized by UV light were taken in a 24-well culture plate and pre-wet with DMEM followed by culturing with BMSCs in cell culture medium at a seeding density of 3 104 cells/scaffold. Cells were seeded to scaffolds in both vertical and horizontal positions and cultured in 5% CO2 environment at 37 C with regular replacement of fresh medium every two days. Morphology of the cells around the scaffold surface and interior was analyzed using a SEM (S-3000N, Hitachi Ltd., Tokyo, Japan). 2.6.3. Mono-Culture with BMSCs and Chondrocytes Qualitative assessment on the viability of adhered BMSCs and chondrocytes in C and V scaffolds were evaluated through a Live/Dead viability/cytotoxicity assay kit (Molecular Probes, Eugene, OR, USA). BMSCs were seeded in pre-wet cylindrical C scaffolds at a seeding density of 3 104 cells/scaffold, whereas chondrocytes were seeded in V scaffolds at the same seeding density and cultured up to 28 days. C scaffolds were cultured in osteogenic medium (OM) (DMEM with 50 l-ascorbic acid phosphate, 0.1 dexamethasone, 10 mM glycerol 2-phosphate, 1% antibiotic-antimycotic, and 10% FBS), whereas V scaffolds were cultured in chondrocyte medium (DMEM/F12 supplemented with 10% FBS and 1% antibiotic-antimycotic). The culture medium was replaced with fresh medium every 2 days and washed with PBS prior to staining. The Live/Dead staining solution was prepared by mixing 2 M calcein AM (excitation 494 nm and emission 517 nm for live cells) with 5 M of ethidium homodimer-1 (EthD-1) (excitation 528 nm and emission 617 nm for dead cells) in culture medium. Samples were incubated with the staining solution at 37 C for 30 min and imaged under a Zeiss LSM 510 Meta confocal laser scanning microscope (Carl Zeiss AG, Jena, Germany). 2.6.4. Cell Proliferation Cylindrical-shaped V, C, and OC scaffolds were sterilized by UV light for 4 h and placed in a GDC-0941 kinase activity assay 24-well culture plate. All scaffolds were pre-wet with DMEM followed by seeding with BMSCs at a density of 1 1 104 cells/scaffold and the cells were allowed to adhere at 37 C for 4 h. After the incubation period, the scaffolds were transferred to a new culture plate containing 1 mL OM and placed in a 37 C humidified 5% CO2 incubator. The cell number was determined by DNA assay using Hoechst 33258 [38]. 2.6.5. Co-Culture with BMSCs and Chondrocytes Cylindrical-shaped OC scaffolds with nHAP-containing bone part were designed to be cultured with BMSCs followed by culturing chondrocytes in the cartilage part. Briefly, the bone part GDC-0941 kinase activity assay of OC scaffolds was rinsed in OM followed by seeding with BMSCs (2 104 cells/scaffold), and maintained in OM for 7, 14, and 21 days. At each time period, the OM was removed and the cartilage part of the OC scaffold was seeded with chondrocytes (1 104 cells/scaffold) and maintained for another 7, 14, and 21 days in chondrocyte medium. Thus, the OC scaffold was immersed in OM and chondrocyte medium respectively before and after chondrocyte seeding in the cartilage part. The respective morphology of BMSCs and chondrocytes in the bone and cartilage parts of OC scaffolds were monitored through SEM. Morphology of BMSCs in the bone part of OC scaffolds after each co-culture time point was further evaluated through SEM observation to verify the cell behavior in bone part with additional culture in chondrocyte medium. 2.6.6. Alizarin Red and Alcian Blue Staining The effect of various stimulating factors in tissue-specific HESX1 cell differentiation was analyzed through Alizarin red (AR) staining of the bone part and Alcian blue (AB) staining of the cartilage part. AR staining was done for both mono- and co-cultured samples, while AB staining was performed only for co-culture. In mono-culture, disc-shaped scaffolds (V, C, and OC) were seeded with BMSCs (1 104 cells/scaffold) and cultured in normal medium (NM, DMEM with GDC-0941 kinase activity assay 10% FBS and 1% antibiotic-antimycotic) and OM. Acellular scaffolds were considered as controls for comparative evaluation towards bone formation. After 14 days culture in both NM and OM, samples were rinsed thrice with PBS and fixed with glutaraldehyde solution (2.5%) for 2 h. 0.5 g Alizarin red S (ARS) was dissolved in 25 mL deionized water GDC-0941 kinase activity assay adjusted to pH 4.1~4.3 with ammonium hydroxide to get the AR staining solution and each scaffold was immersed in 1 mL of the same, followed by incubation for 1 h at room temperature..