Comparison of bilayered structures of gellan gum with and without incorporation of gold nanorods for osteochondral tissue engineering

Abstract

Event Abstract Back to Event Comparison of bilayered structures of gellan gum with and without incorporation of gold nanorods for osteochondral tissue engineering Raphael Canadas1, 2, Silvia Vieira1, 2, Stephanie Vial1, 2, Alexandra P. Marques1, 2, Rui L. Reis1, 2 and Joaquim M. Oliveira1, 2 1 University of Minho, 3B´s Research Group – Biomaterials, Biodegradables and Biomimetics, Portugal 2 University of Minho, ICVS/3B's - PT Government Associate Laboratory, Portugal Introduction: In orthopedics, the regeneration and repair of osteochondral defects after trauma, cancer, or metabolic disorders is still a major clinical challenge, usually leading to an osteoarthritic condition which is a major cause of disability during aging. By the age of 60, close to 100% of the population will have degeneration of knee cartilage[1]. In order to outperform current methods, novel tissue engineering approaches have been proposed to take advantage of the use of stem cells, growth factors and/or pre-programming the stem cells fate, bioreactors and multilayered scaffolds design. The need for simultaneous regenerate both cartilage and subchondral bone in the same scaffold, is a requirement in osteochondral defects. The incorporation of strategic cues to guide the cell fates towards chondro- and osteo-genesis, is an approach to be considered to regenerate both tissues. A previous study performed in our Group showed that the presence of gold nanorods may potentiate the effect of the osteogenic media towards osteocyte-like phenotype, with concomitant reduction of ALP activity and increased mineralization capacity[2]. In the herein presentes work gellan gum based multilayered structures were developed to recreate the bone, calcified cartilage and hyaline cartilage regions. Gold nanorods (AuNRs) and hydroxyapatite particles were incorporated as it is hypothesized its benefits for promoting the osteogenic differentiation of human fat pad adipose-derived stem cells. Materials and Methods: Bilayered sponge-like scaffolds were developed to act as a template for culturing human fat pad adipose-derived stem cells (hASCs). Bilayered spongy structures made of low acyl gellan gum (LAGG)-LAGG/hydroxyapatite (HAp) with and without incorporation of AuNRs were produced integrating cartilage- and bone-like layers (figure 1 represents the AuNRs formation and crosslinking with LAGG). The effect of AuNRs was evaluated and characterized for hASCs osteogenic differentiation following standard protocols. Likewise, the chondrogenic differentiation of hASCs in LAGG was assessed. Figure 1. AuNRs formation, with incorporation of cetyltrimethylammonium bromide (CTAB), poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) and further crosslinking with LAGG. Results and Discussion: The freeze-dried bilayered scaffolds composed of LAGG2%(w/v)-LAGG2%/HAp30% (w/w) and LAGG/Goldnanorod 0.25mM-LAGG/Gold nanorod 0.25mM/HAp30% (w/w) have a gradient of HAp in the bone-like layer that, unlike cartilage-like layer, presented a bioactive behavior. The bilayered structures possessed about 90% porosity, 500 µm of pore size and 85% interconnectivity as determined by Micro-CT analysis. Swelling and degradation tests revealed that the structures absorb about 120% of their weight and lost 10% of their mass after 30 days in phosphate buffered saline solution (figure 2). Figure 2. Bilayered scaffolds. A) Bilayer hydrogel after crosslinking in calcium solution and prior freeze-drying; B) X-ray and micro-CT (C,D) visualization of the structure of LAGG with HAp particles in the bone-like layer; E) Alizarin red staining of HAp particles. In vitro studies with isolated hASCs from Fat Pad are being performed to compare the osteogenic and chondrogenic outcomes in the bilayered structures and the effect of the AuNRs regarding alkaline phosphatase (ALP) activity and mineralization. Conclusions: The described structures present the potential for orthopedic applications, namely for osteochondral tissue regeneration. Although the incorporation of AuNRs was already proved to promote the mineralization process, the mechanism seems to be an independent pathway from ALP activity and is now under investigation in this work. The incorporation of the gold nanorods changed the hydrophilicity of the structure, which can be useful also for the calcified cartilage transition zone, since cartilage present a high hydrated phenotype. This study received funding from Portuguese Foundation for Science and Technology (FCT) through the project OsteoCart (PTDC/CTM-BPC/115977/2009) and from European Union’s Seventh Framework Programme (FP7/2007-2013) for REGPOT-CT2012-316331-POLARIS. The authors also thanks FCT for the PhD scholarship provided to R. F. Canadas (SFRH/BD/92565/2013).