Abstract
Ceramic and metallic nanoparticles can improve the mechanical and biological properties of polymeric scaffolds for bone tissue engineering (BTE). In this work, nanohydroxyapatite (nHAp) and nano-copper-zinc alloy (nCuZn) were added to a chitosan/gelatin (Ch/G) scaffold in order to investigate the effects on morphological, physical, and biocompatibility properties. Scaffolds were fabricated by a freeze-drying technique using different pre-freezing temperatures. Microstructure and morphology were studied by scanning electron microscopy (SEM), glass transition (Tg) was studied using differential scanning calorimetry (DSC), cell growth was estimated by MTT assay, and biocompatibility was examined in vitro and in vivo by histochemistry analyses. Scaffolds and nanocomposite scaffolds presented interconnected pores, high porosity, and pore size appropriate for BTE. Tg of Ch/G scaffolds was diminished by nanoparticle inclusion. Mouse embryonic fibroblasts (MEFs) cells loaded in the Ch/G/nHAp/nCuZn nanocomposite scaffold showed suitable behavior, based on cell adhesion, cell growth, alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation, and histological in vitro cross sections. In vivo subcutaneous implant showed granulation tissue formation and new tissue infiltration into the scaffold. The favorable microstructure, coupled with the ability to integrate nanoparticles into the scaffold by freeze-drying technique and the biocompatibility, indicates the potential of this new material for applications in BTE.
Highlights
Bone substitutes are becoming a suitable option to reconstruct bone defects instead of autogenous or allogenous bone graft procedures [1]
The scanning electron microscopy (SEM) analysis of the scaffolds prepared without nanoparticles at different pre-freezing temperatures revealed a 3D microstructure with pores of different sizes and shapes (Figure 1a, upper row) including changes in the surface (Figure 1a, lower row)
Gas pycnometry and SEM analyses showed that increasing the pre-freezing temperature during scaffold preparation produced an increase of porosity from 97.8 to 99.5% and the pore size from 113 to 143 μm respectively (Figure 1b)
Summary
Bone substitutes are becoming a suitable option to reconstruct bone defects instead of autogenous or allogenous bone graft procedures [1]. A requirement for bone tissue engineering substitutes is the development of three-dimensional scaffolds that are biocompatible, biodegradable, and osteoinductive. They must support cell adhesion, proliferation, and viability [1,2,3]. A natural copolymer of glucosamine and N-acetylglucosamine [4], has been widely used as scaffold in tissue engineering because it is biocompatible, non-toxic, reabsorbable, and has antimicrobial properties [5]. Gelatin is obtained from the denaturation of collagen and is suitable as a scaffold because is biocompatible, non-toxic, and reabsorbable [6]. Gelatin has low immunogenicity and contains Arg-Gly-Asp (RGD)-like sequences that promote cell adhesion [7,8]
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