Biocompatibility and integrin-mediated adhesion of human osteoblasts to poly(dl-lactide-co-glycolide) copolymers

Abstract

The biocompatibility of polylactic acid (PLA) and polyglycolic acid (PGA) copolymers, employed in manufacturing bone-graft substitutes, is affected by their chemical composition, molecular weight and cell environment, and by the methods of polymerization and processing. Their in vitro bioactivity on human osteoblasts has been investigated very little. We first evaluated the behavior of primary human osteoblasts cultured in close contact with 75:25 and 50:50 PLA–PGA copolymers for 14 days adopting a cell culture system that allowed us to evaluate the influence of direct contact, and of factors released from polymers. The copolymers had no negative influence on cell morphology, cell viability and proliferation. Alkaline phosphatase (ALP) activity and osteocalcin production were also not affected. The initial adhesion of osteoblasts on implant surfaces requires the contribution of integrins, acting as a primary mechanism regulating cell-extracellular matrix (ECM) interactions. We observed that adhesion of osteoblasts to PLA–PGA copolymers, 2 h after plating, was reduced by ≈70% by antibodies capable to block integrin β₁ and α₅β₁ complex and only by ≈30% by an anti-integrin α_v antibody. Therefore, β₁ integrins may represent a predominant adhesion receptor subfamily utilized by osteoblasts to adhere to PLA–PGA copolymers. These materials do not show any negative influence on cell proliferation and differentiation.

Introduction

Polyesters, including polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers (polylactide-co-glycolide (PLGA)) are employed in manufacturing internal devices (Athanasiou et al., 1996, Athanasiou et al., 1998, Leenslag et al., 1987, Zislis et al., 1989, Schakenraad and Dijkstra, 1991). The biocompatibility of a biodegradable polymer is affected by its chemical composition, molecular weight, and crystallinity and by the cell environment where it has been placed (Park and Cima, 1996, Ikarashi et al., 2000a). A loss of biocompatibility of these polymers has been related to their degradation rate (Ignatius and Claes, 1996). Schakenraad et al. (1989), for instance, observed a more intense tissue reaction for polymers containing PLA, with higher degradation rates. This process ultimately leads to the release not only of degradation products, but also of residual monomers, catalysts and additives (Taylor et al., 1994). Moreover, degradation of PLA and PLGA polymers may be influenced by other factors, including polymer characteristics, the methods of polymerization and processing, and sterilization and storage conditions (Vert et al., 1992, Vert et al., 1994).

Possible interactions between bone cells and biomaterial should be evaluated in cell culture systems in order to see how osteogenic cells respond to these materials (for a review see Morrison et al., 1995). In vitro biocompatibility of PLA and PLGA polymers has been mainly evaluated in immortalized cell lines such as mouse fibroblasts or osteogenic cells (Elgendy et al., 1993, Morrison et al., 1995, Schwartz et al., 1999). However, in these models several cellular processes such as proliferation, apoptosis and expression of differentiation markers may be modified (Puleo et al., 1991, Xynos et al., 2000, Ikarashi et al., 2000b).

In this study we investigated the behavior of primary human osteoblasts cultured in the presence of PLGA copolymers for 14 days. We adopted a cell culture system that has been employed to evaluate the influence of direct contact and of factors released from bone-graft substitutes (Winn and Hollinger, 2000). To evaluate biocompatibility we assessed cell viability, proliferation and morphology. We also measured the alkaline phosphatase (ALP; E.C. 3.1.3.1) and osteocalcin. ALP is involved in preparing the extracellular matrix (ECM) for the ordered deposition of mineral and serves as an early marker of differentiation for osteoblasts (Attawia et al., 1995). Osteocalcin is the most abundant non-collagenous protein of the mineralized extracellular bone matrix, and increases during late osteoblastic differentiation (Desbois and Karsenty, 1995).

Events leading to integration of polymeric materials into bone take place primarily at the polymer interface and require different factors (for a review see Ziats et al., 1988, Puleo and Nanci, 1999). These include the initial cell adhesion and spreading over implant surfaces through extracellular matrix proteins, such as fibronectin, vitronectin, fibrinogen and collagen (Buck and Horwitz, 1986, Grzesik and Robey, 1994, Sinha and Tuan, 1996, Chang et al., 1998). In fact, adhesive cells utilize ECM proteins to attach and migrate on substrates, exchange signals that block apoptosis and allow cell cycle progression and the appearance of tissue-specific phenotypes (Garcia et al., 1999, Zhu et al., 1996, Cutler and Garcia, 2003). Cell adhesion to ECM proteins is primarily mediated by integrins, transmembrane receptors composed of α and β subunits, with at least 12 different α and nine β subunits described to date (for a review see Ivaska and Heino, 2000, van der Flier and Sonnenberg, 2001). Upon ligand binding, integrins cluster together and organize into complexes that contain structural and signaling proteins (Jockusch et al., 1995, Burridge and Chrzanowska-Wodnicka, 1996, Damsky, 1999, Stephansson et al., 2002). Human osteoblasts grown on tissue culture polystyrene express primarily the integrin subunits α₁₋₆, α_v and β_1,3 (Hughes et al., 1993, Gronthos et al., 1997, Bennett et al., 2001). Integrin expression appears to be modified in osteoblasts grown on surfaces such as titanium and cobalt-chrome or on biodegradable polymers such as PLA and PLGA (Gronowicz and McCarthy, 1996, Geissler et al., 2000, El-Amin et al., 2002). The contribution of any specific integrin to osteoblast adhesion to metal materials has been recently evaluated by functional blocking antibodies (Krause et al., 2000, Matsuura et al., 2000, Schneider et al., 2001, Zreiqat et al., 2002, Cutler and Garcia, 2003); however, to date, little is known about their involvement in cell adhesion to PLGA. Therefore, in the second part of this study we evaluated osteoblast adhesion to PLGA copolymers in the presence of antibodies capable to block integrins β₁, α₅β₁ and α_v.