Does seeding density affect in vitro mineral nodules formation in novel composite scaffolds?

This study investigated the human alveolar osteoblasts (AOs) proliferation and extracellular matrix formation at seeding density of 0.05, 0.1, 0.2, 0.4, and 0.8 million (M) per 3x4x4 mm3 on medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) scaffolds designed for bone regeneration. Over 80-90% of the initial seeded cells were retained in the scaffolds after 24 h. AOs bridged over pores at density of 0.2M/scaffold and below, but formed cell balls at density of 0.4M/scaffold and above. At seeding density of 0.2M and below, cell proliferation increased with time having DNA content peaked to 1600 ng/scaffold at day 21 and 28, respectively, whereas at 0.4 and 0.8M, the corresponding DNA content decreased to 1600 ng in 28 days. At day 7, higher alkaline phosphatase (ALP) activity and higher osteocalcin (OCN) secretion were detected at 0.2M/scaffold and below. After 28 days, multilayered cell-sheet formation and collagen fibers were observed at all densities. ALP and OCN in matrix and mineral nodules were found mainly at the border of AOs-scaffold construct. These findings demonstrated that the density of 0.2M and below per 3 x 4 x 4 mm(3) scaffold resulted in better cell proliferation and extracellular matrix synthesis, potentially resulting in better mineralized tissue formation.