Human Osteoprogenitor Growth and Differentiation on Synthetic Biodegradable Structures After Surface Modification X. B. YANG, H. I. ROACH, N. M. P. CLARKE, S. M. HOWDLE, R. QUIRK, K. M. SHAKESHEFF, and R. O. C. OREFFO University Orthopaedics, University of Southampton, Southampton General Hospital, Southampton, UK School of Pharmaceutical Sciences, University Park, Nottingham, UK 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 con- firmed with alkaline phosphatase activity as well as immu- nocytochemistry 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 pre- senting adsorbed fibronectin (FN). Both modifications en- hanced 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 ascor- bate-2-phosphate for 4 – 6 weeks, extensive osteoblast im- pregnation was observed by confocal and fluorescence mi- croscopy. Cell viability in extended culture was maintained as analyzed by expression of Cell Tracker green and negli- gible ethidium homodimer-1 (a marker of cell necrosis) stain- ing. Alkaline phosphatase activity, type I collagen, Cbfa-1, and osteocalcin expression were observed by immunocyto- chemistry. Mineralization of collagenous matrix took place after 4 weeks, which confirmed the expression of the mature osteogenic phenotype. These observations demonstrate suc- cessful adhesion and growth of human osteoprogenitors on protein- and peptide-coupled polymer films as well as migra- tion, expansion, and differentiation on three-dimensional bio- degradable PLGA scaffolds. The use of peptides/proteins and three-dimensional structures that provide positional and en- vironmental information indicate the potential for biomi- metic structures coupled with appropriate factors in the development of protocols for de novo bone formation. (Bone 29:523–531; 2001) © 2001 by Elsevier Science Inc. All rights reserved. Key Words: Human; Osteoprogenitor; Bone marrow; Biode- gradable polymer; Poly(-lactic-co-glycolic acid) (PLGA); Tis- sue engineering. Introduction A desirable clinical orthopedic requirement is a biodegradable biomimetic material that induces and promotes significant new bone formation by osteogenic cells at a required site; for exam- ple, around metal implants after revision surgery, in fracture gaps, or within osteoporotic bone. Traditional approaches have involved the use of autogenous or allogenic bone. 28 However, limited availability (autogenous bone) or tissue rejection (allo- genic bone) has resulted in the search for other methods to generate new bone for skeletal use. 26,27 Furthermore, the current approaches for bone regeneration do not take in account the biological cues that are necessary to mimic the bone cell-bone matrix interactions within the bone microenvironment. A prom- ising approach is the development of biomimetic materials, which provide microenvironments for cell-matrix interactions that mimic biological environments. 33 The development and use of synthetic materials, such as poly(lactic acid) (PLA), poly(-lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polyanhydrides, polyimides, polypho- sphazenes, or collagen, as scaffolds for cell transplantation and tissue growth has made the development of such biomimetic scaffolds a realistic objective. 6,33,37 These synthetic materials, approved and widely used in the formation of resorbable sutures, scaffolds, and drug-delivery devices, are: (i) biocompatible; (ii) processable into a three-dimensional structure; and (iii) eventu- ally degradable. In recent years, procedures for the surface modification of these materials, with biological agents, have been developed (reviewed in Shakesheff et al. 40 ). Thus, an alternative approach for skeletal repair is the selection, expan- Address for correspondence and reprints: Dr. Richard O. C. Oreffo, University Orthopaedics, MP817, University of Southampton, Southampton General Hospital, Southampton S016 6YD, UK. E-mail: roco@soton.ac.uk Bone Vol. 29, No. 6 December 2001:523–531 523 © 2001 by Elsevier Science Inc. 8756-3282/01/$20.00 All rights reserved. PII S8756-3282(01)00617-2