Culture and Characterization of Osteoblasts from the Parietal Bone of Neonatal Wistar Rats
Abstract
The proposed protocol is a simple, economical, and efficient method for primary osteoblast culture, demonstrating greater stability and yields than traditional approaches. Osteoblast in vitro cultures are widely used as a tool for studying bone formation and regeneration. However, protocol execution is one of the most challenging and least standardized steps in osteoblast culture. Objective: to present a modified, useful, and simple protocol for osteoblast culture from the parietal bone of Wistar rats. Methods: Parietal bone from 11-day-old Wistar rats was used; the sample was fractionated for histochemical analysis and cell culture, while the human osteoblast cell line (NHost) was seeded as a control. Phenotypic characterization of osteoblasts in both cultures was performed by immunostaining for alkaline phosphatase and osteocalcin. Primary cultures were stained with alizarin red for morphological and calcium deposition characterization. Results: Parietal bone was found to undergo intramembranous ossification with higher levels of osteoblasts in the suture periosteal area. Positive immunostaining for the three markers was observed in both primary and control cultures. After culture confluence, osteoblasts form three-dimensional structures that resemble bone spicules. Conclusions: Preservation of the periosteum and sutures during bone collection is crucial because these regions contain abundant osteoblastic progenitors. The proposed model can be a valuable tool for tissue engineering and bone biology research, particularly in low-resource settings where the high costs of more complex techniques may be a barrier.
Keywords: Calvaria; cell culture; osteoblast; periosteum; Wistar rat.
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