(173e) Osteoblast Response to Amorphous Calcium Phosphate in Vitro

Engineered bone tissue is an attractive means for the repair of bone defects resulting from trauma, disease, or congenital abnormalities. Calcium phosphates, such as hydroxyapatite (HAP), have been widely used as bone substitutes because of their biocompatibility and osteoconductivity. However, HAP lacks osteoinductive properties to stimulate osteogenesis and is resistant to biodegradation. The release of osteoinductive factors during degradation of a calcium phosphate would offer greater potential for the stimulation of osteogenesis.

Amorphous calcium phosphate (ACP) is a bioactive ceramic that is soluble and converts to HAP under aqueous conditions. During this conversion, Ca²⁺ and PO₄^3- ions are transiently released into the surrounding environment. We predict that the release of these ions in the vicinity of osteoprogenitor cells will stimulate osteoblast differentiation. Furthermore, a biodegradable polymer scaffold containing ACP would provide the osteoconductive and osteoinductive properties necessary to stimulate osteogenesis and facilitate healing of a bony defect.

To determine the effects of ACP in tissue culture, mineral was added to growth medium and maintained in a 37°C, 5% CO₂, 95% relative humidity incubator for 48 hours. Samples of medium were collected at 0.5, 1, 2, 4, 24, 48 hours for pH and Ca²⁺ and PO₄^3- concentration measurements. Remaining mineral was collected and analyzed by X-ray diffraction to assess conversion of ACP to HAP.

The effect of ACP on osteoprogenitor cells was assessed by adding ACP particles to cell layers at different stages of cell proliferation and differentiation. Rat bone marrow stromal cells (BMSC) were cultured for 21 days in osteogenic medium and mineral was added to cell layers at 4, 11, 18 days. β-glycerophosphate was excluded from osteogenic medium to allow for more rigorous assessment of the effect of ion release from ACP. After 21 days of culture, western blot analysis and quantitative real time PCR were utilized to quantify the synthesis of osteogenic markers and evaluate the effect of ACP on BMSC differentiation.