Yang, X.B., Roach, H.I., Clarke, N.M.P., Howdle, S.M., Quirk, R., Shakesheff, K.M. and Oreffo, R.O.C.
Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification
Bone, 29, (6), . (doi:10.1016/S8756-3282(01)00617-2).
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The ability to generate new bone for skeletal use is a major clinical need. Biomimetic scaffolds that interact and promote osteoblast differentiation and osteogenesis offer a promising approach to the generation of skeletal tissue to resolve this major health-care issue. In this study we examine the ability of surface-modified poly(lactic acid) (PLA) films and poly(lactic-co-÷glycolic acid) (PLGA) (75:25) porous structures to promote human osteoprogenitor adhesion, spreading, growth, and differentiation. Cell spreading and adhesion were examined using Cell Tracker green fluorescence and confocal microscopy. Osteogenic differentiation was confirmed with alkaline phosphatase activity as well as immunocytochemistry for type I collagen, core binding factor-1 (Cbfa-1), and osteocalcin. Poor cell growth was observed on nonmodified PLA films and PLGA scaffolds. The polymers were then coupled with RGD peptides [using poly(L-lysine), or PLL] and physical adsorption as well as PLA films presenting adsorbed fibronectin (FN). Both modifications enhanced cell attachment and spreading. On PLA-FN and PLA-PLL-GRGDS films, the osteoblast response was dose dependent (20 pmol/L to 0.2 ?mol/L FN and 30 nmol/L to 30 ?mol/L PLL-GRGDS) and significant at concentrations as low as 2 nmol/L FN and 30 nmol/L PLL-GRGDS. With optimal concentrations of FN or RGD, adhesion and cell spreading were comparable to tissue culture plastic serum controls. In PLGA (75:25) biodegradable porous scaffolds, coated with FN, PLL-GRGDS, or fetal calf serum for 24 h in ?MEM alone, prior to growth in dexamethasone and ascorbate-2-phosphate for 4–6 weeks, extensive osteoblast impregnation was observed by confocal and fluorescence microscopy. Cell viability in extended culture was maintained as analyzed by expression of Cell Tracker green and negligible ethidium homodimer-1 (a marker of cell necrosis) staining. Alkaline phosphatase activity, type I collagen, Cbfa-1, and osteocalcin expression were observed by immunocytochemistry. Mineralization of collagenous matrix took place after 4 weeks, which confirmed the expression of the mature osteogenic phenotype. These observations demonstrate successful adhesion and growth of human osteoprogenitors on protein- and peptide-coupled polymer films as well as migration, expansion, and differentiation on three-dimensional biodegradable PLGA scaffolds. The use of peptides/proteins and three-dimensional structures that provide positional and environmental information indicate the potential for biomimetic structures coupled with appropriate factors in the development of protocols for de novo bone formation.
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