Development of a 3D Collagen-Hydroxyapatite Composite in vitro Culture Model for Osteoblasts and Osteocytes
Osteoporosis is a disease characterized by a severe loss in bone mineral density, putting individuals at elevated risk of bone fracture. The disorder is extremely prevalent, with high associated costs. Pharmaceutical treatments exist, but currently have severe drawbacks, and preventative measures are eﬀective primarily within an individual's ﬁrst two decades of life, long before the normal age of diagnosis. To better develop treatments in a high throughput physiological setting, an in vitro model for bone cell culture that accurately recapitulates the in vivo cellular environment is needed. Thus, a 3D culture system has been developed utilizing porcine skin collagen oligomers (PSC) and precipitated hydroxyapatite (HA) nanoparticles to support de novo bone formation and diﬀerentiation of osteoblasts into osteocytes. Cells isolated from DMP1-Cre x mT/mG mice were cultured in 3D matrices with diﬀering concentrations of PSC and HA for 3 and 56 days in osteogenic medium, before being sampled for testing. Fixed culture sections were imaged using confocal microscopy to quantify diﬀerentiation of osteoblasts to osteocytes, indicated by the shift from cellular expression of red ﬂuorescent protein to green ﬂuorescent protein through the activation of the promoter for the osteocyte-speciﬁc dentin matrix protein 1. These data demonstrated a signiﬁcant proportional increase in late osteoblasts and and osteocytes. µCT analysis showed a signiﬁcant increase in mineralization of the ECM, exclusive to cultures initially formed with HA. This was reﬂected by increased mechanical stiﬀness at high strain in some culture conditions.
Main, Purdue University.
Cellular biology|Biomedical engineering
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