Layer-by-Layer assembled growth factor reservoirs for steering the response of 3T3-cells

Highlights

• Reservoirs for fibroblasts growth factors (FGF) using (Heparin/Chitosan)_n multilayers are efficient to store aFGF and bFGF for long periods.

• These reservoirs are able to improve the proliferation of NIH/3T3 fibroblasts.

• Moreover, they are very useful as coatings to make FGFs available at biomaterial surfaces.

Abstract

Layer-by-Layer (LbL) assemblies of heparin (Hep) and chitosan (Chi) were prepared for use as reservoirs for acidic and basic fibroblast growth factors (aFGFs and bFGFs, respectively). The effects of the architecture and composition of the reservoirs on the viability and proliferation of NIH-3T3 fibroblast cells were studied under starvation conditions. The reservoir stability was monitored by ellipsometry. The aFGF and bFGF loadings were determined using a dissipation-enhanced quartz crystal microbalance (QCM-D). Stability and release assays were performed in a phosphate buffer at physiological conditions. The results demonstrated that the amount of aFGF and bFGF loaded into and released from LbL reservoirs composed of 3 and 6 layer pairs could be controlled. Cell culture assays in low serum culture medium (LSCM) demonstrated that incorporating very small amounts of aFGF and bFGF into the (Hep/Chi)_n multilayers significantly improved the proliferation of the NIH-3T3 fibroblasts. The cells did not proliferate on (Hep/Chi)_n assemblies prepared in the absence of FGF under identical conditions. The LbL reservoirs were highly effective for the long-term storage (up to 9 months) of aFGF and bFGF. This work demonstrates the potential of LbL reservoirs for use as biomaterial coatings.

Introduction

Signaling molecules such as growth factors must be delivered to their target cells in a spatio-temporally controlled fashion to ensure that their specific bioactivity for a given cell type is fully utilized [1]. Although many signaling molecules control the formation of three-dimensional multicellular natural tissues during organogenesis, current tissue engineering approaches often involve the introduction of growth factors via simple scaffold-based delivery systems with very limited control over local concentrations or release kinetics. To engineer complex tissues, three-dimensional patterned architectures in which compartments (reservoirs) containing different signaling molecules are separated by walls (barriers) with adjustable permeability are ideal. The Layer-by-Layer (LbL) assembly method has been used to fabricate different reservoir/barrier architectures containing biomolecules. In this work, two-dimensional, single-component reservoirs for acidic and basic fibroblast growth factors (aFGFs and bFGFs, respectively) were fabricated by LbL assembly, and the effects of the reservoirs on 3T3 cells cultured under starvation conditions were studied.

Layer-by-Layer assembly is a highly versatile method for preparing multi-component coatings on different surfaces [2], [3]. Efficient methods for fabricating multilayer films containing biomolecules for biomedical and biomaterial applications have been widely studied [4], [5].

Fibroblast growth factors are polypeptide-signaling molecules with a molar mass ranging from 16 to 34 kDa. These molecules, which were discovered by Armelin in 1973, support cell growth and division [6]. FGFs are well known for their role in cell development [7], proliferation, organogenesis (see above), cell differentiation, cell migration [6], [8], [9], wound healing [10], [11], and angiogenesis [12], [13]. The release of sufficient amounts of FGFs can induce progenitor cell recruitment without the need for stem cell implantation [14].

Systems with FGFs incorporated into polyelectrolyte multilayers have been reported to mimic physiological conditions and to induce wound healing and tissue regeneration. FGF-containing multilayers have been assembled on flat [14], [15], [16], [17], [18] and spherical surfaces [19], [20] and have also been incorporated into polyelectrolyte multilayers to improve the compatibility of biomaterials. However, FGFs are very labile and can lose their activity in less than one week when stored in solution at 2–8 °C. Freeze-thaw cycles can also cause FGF inactivation. In this work, FGFs were incorporated into multilayers prepared by LbL assembly to improve their stability in long-term storage. Specifically, (heparin/chitosan)_n ((Hep/Chi)_n) multilayers with different architectures and FGF contents were used as growth factor reservoirs and delivery systems for cultured cells.

The model proteins employed in this study were acidic FGF (aFGF, isoelectric point pI = 5.6) and basic FGF (bFGF, pI = 9.6). It is known that aFGF can induce mitosis, cell migration, and cell differentiation in most mesodermal cells. It also influences angiogenesis and regulates many other biological responses [13], [21]. bFGF belongs to a family of proteins that stimulate fibroblast proliferation [15] and activate angiogenesis, chemotaxis, and periodontal ligament proliferation [22].

Heparin is a polysulfated glycosaminoglycan with a high polydispersity and large variations in its saccharide monomer sequence. It has the highest negative charge density of any known biological molecule. The assembly of heparin molecules with FGFs can induce conformational changes in the FGFs, thereby improving their resistance to thermal and enzymatic denaturation; for example, the inactivation of bFGF is reduced at acidic pHs [23]. Chitosan is a naturally derived polycation that has been extensively studied for its numerous positive biological properties [24]. It is positively charged at low pH and can be solubilized in aqueous media at pH 4.5 or lower. It has been categorized as “generally recognized as safe” (GRAS) by the U.S. Food and Drug Administration.