ABSTRACT
Although hydroxyapatite (HAP) and ß-tricalcium phosphate have been used extensively as osteoconductive minerals in biomaterial scaffolds for bone regeneration, they lack the capacity to stimulate osteoblastic differentiation of progenitor cells. In contrast, amorphous calcium phosphates (ACPs), which convert to HAP under aqueous conditions, have the potential to facilitate osteoblastic differentiation through the transient local release of calcium and phosphate ions. Therefore, in this study ACPs were synthesized using zinc and zirconia divalent cations as stabilizers (denoted ZnACP and ZrACP, respectively) and compared to HAP. Analysis of ion release into serum-containing cell culture medium revealed transiently elevated levels of calcium and phosphorous, consistent with the enhanced solubility of ZrACP and ZnACP relative to HAP. In addition, X-ray diffraction analysis revealed partial conversion of ZrACP to HAP but no conversion of ZnACP after 96 h. Next, scaffolds were fabricated by sintering mixtures of 300-500 µm poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres and 0.5 wt% calcium phosphate mineral (HAP, ZrACP or ZnACP) at 70 °C for 24 h. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate particulates clinging to the microsphere surfaces both prior to and after 14 days in culture medium. Finally, the incorporation of calcium phosphate resulted in a lower compressive modulus in the range 127 to 74-89 MPa. Taken together, these results indicate that ZrACP, ZnACP and HAP minerals exhibit very different properties, and therefore may elicit different osteoblastic responses in vitro.
Subject(s)
Calcium Phosphates/chemistry , Lactic Acid/chemistry , Polyglycolic Acid/chemistry , Tissue Engineering/methods , Animals , Crystallization , Culture Media/chemistry , Humans , Ions , Kinetics , Microscopy, Electron, Scanning/methods , Microspheres , Osteoblasts/cytology , Polylactic Acid-Polyglycolic Acid Copolymer , Porosity , Pressure , Surface Properties , Tissue Scaffolds/chemistry , X-Ray Diffraction , Zinc/chemistry , Zirconium/chemistryABSTRACT
Calcium phosphate-decorated polyester matrices are promising scaffolds for bone tissue engineering that combine the tunable degradation of synthetic polymers and the osteoconductivity of calcium phosphate minerals. In this study, scaffolds decorated with stabilized amorphous calcium phosphate (ACP) minerals-which exhibit sustained dissolution and release of calcium and phosphate ions-were tested for their ability to support osteoblast proliferation and stimulate differentiation. The two ACPs tested were zirconia-hybridized ACP (ZrACP), which releases ions over a few days and converts in aqueous solution to hydroxyapatite (HAP), and zinc-hybridized ACP (ZnACP), which has a longer period of sustained ion release. MC3T3-E1 pre-osteoblasts were cultured in these scaffolds for up to 21 days, and cell number, alkaline phosphatase (ALP) activity and expression of osteogenic and bone-specific proteins were measured. Cell number, prostaglandin E(2) (PGE(2) ) synthesis and osteopontin (OPN) mRNA expression were elevated on calcium phosphate-decorated scaffolds relative to PLGA controls, while mRNA expression of osteocalcin (OCN), bone sialoprotein and bone morphogenetic protein (BMP)-4 were suppressed. Although MC3T3-E1 responses to the two ACPs were not statistically different, ZrACP-which converts more quickly to HAP-gave rise to slightly higher levels of mRNA expression for BMP-4, osterix, vascular endothelial growth factor (VEGF)-A, OCN and OPN, but slightly lower levels of PGE(2) synthesis, ALP activity and cell number. These results indicate that sintered PLGA microsphere scaffolds decorated with 0.5 wt% ZnACP or ZrACP support cell attachment and elicit a series of biological responses, but these responses do not appear to accelerate osteoblast differentiation.
Subject(s)
Calcium Phosphates/chemistry , Cell Differentiation , Lactic Acid/chemistry , Osteoclasts/cytology , Polyglycolic Acid/chemistry , Tissue Scaffolds/chemistry , Animals , Antigens, Differentiation/biosynthesis , Cell Line , Gene Expression Regulation , Mice , Microspheres , Osteoclasts/metabolism , Polylactic Acid-Polyglycolic Acid CopolymerABSTRACT
Amorphous calcium phosphates (ACPs) are attractive fillers for osseous defects and are stabilized through the incorporation of transition metals such as zirconium and zinc. As ACP converts in solution to hydroxyapatite (HAP) in a manner marked by a transient release of calcium and phosphate ions, it is capable of stimulating osteoblastic differentiation. Zinc is known to retard ACP conversion to HAP, and--when incorporated into ceramic biomaterials--has been shown to stimulate osteoblastic differentiation. Because zinc deficiency in vivo is marked by skeletal defects, we postulated that zinc ions released from ACP and other minerals could stimulate proliferation and osteoblastic differentiation of progenitor cells. To test this hypothesis, rat bone marrow stromal cells were cultured in osteogenic medium containing basal (3 x 10(-6) M) or supplemented Zn(2+) concentrations (1 x 10(-5) and 4 x 10(-5) M) for up to 3 weeks. No significant effects of zinc concentration on cell number, alkaline phosphatase activity, total protein content, collagen synthesis, or matrix mineralization were found.
