Characterization of Cell Differentiation on CPC for Bone Tissue Engineering

Calcium phosphate cement (CPC) is osteoconductive and moldable, can conform to complex cavity shapes and set in-situ to form hydroxyapatite. OBJECTIVES: To quantify the differentiation of MC3T3-E1 preosteoblast cells cultured on CPC disks containing chitosan lactate and bone morphogenic protein-2 (BMP-2). METHODS: CPC (a mixture of tetracalcium phosphate and dicalcium phosphate anhydrous at a 1:1 molar ratio) was mixed at a powder:liquid ratio of 3:1 with either water (traditional CPC), or 15% chitosan solution (reinforced CPC). BMP-2 was added to the chitosan solution at 2 µg BMP-2 per sample. Disk molds were filled with CPC paste and incubated at 37 °C in a humidified environment for 48 h. 1.5 mL of osteogenic differentiation medium containing 10 mM b-glycerophosphate, 50 µg/mL ascorbic acid and 20,000 cells/mL were added to each sample. After 14 d, differentiation was quantified by measuring alkaline phosphatase (ALP) activity and normalizing the data to the DNA concentration, resulting in units of (pNPP mM/min)/(DNA (µg)). RESULTS: The normalized ALP activity (mean ± sd; n = 5) of cells seeded on CPC with water as liquid and no BMP-2 was (33 ± 7). The addition of chitosan increased the ALP activity to (285 ± 38) (p < 0.05). The further addition of BMP-2 to the CPC-chitosan composite increased the ALP activity to (378 ± 128). All of the cells cultured on CPC composites exhibited enhanced ALP activity when compared with tissue culture polystyrene (TCPS) control (12 ± 4) (p < 0.05). CONCLUSION: MC3T3-E1 preosteoblasts showed significantly enhanced osteogenic differentiation on reinforced CPC with growth factor BMP-2 when compared with TCPS control and traditional CPC control. This moldable, in-situ hardening hydroxyapatite composite with growth factor delivery may have enhanced osteogenic potential for tissue engineering in dental and craniofacial applications. Supported by NIDCR DE14190.