Nanofibrous poly(lactide-co-glycolide) membranes loaded with diamond nanoparticles as promising substrates for bone tissue engineering

Martin Parizek¹, Timothy EL Douglas², Katarina Novotna¹, Alexander Kromka³, Mariea A Brady⁴, Andrea Renzing⁴, Eske Voss⁴, Marketa Jarosova³, Lukas Palatinus³, Pavel Tesarek⁵, Pavla Ryparova⁵, Vera Lisa¹, Ana M dos Santos², Lucie Bacakova^1
1Department of Biomaterials and Tissue Engineering, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; ²Polymer Chemistry and Biomaterials Group, Ghent University, Ghent, Belgium; ³Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; ⁴Department of Oral and Maxillofacial Surgery, University of Kiel, Kiel, Germany; ⁵Czech Technical University in Prague, Faculty of Civil Engineering, Prague, Czech Republic

Background: Nanofibrous scaffolds loaded with bioactive nanoparticles are promising materials for bone tissue engineering.
Methods: In this study, composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA) and diamond nanoparticles were fabricated by an electrospinning technique. PLGA was dissolved in a mixture of methylene chloride and dimethyl formamide (2:3) at a concentration of 2.3 wt%, and nanodiamond (ND) powder was added at a concentration of 0.7 wt% (about 23 wt% in dry PLGA).
Results: In the composite scaffolds, the ND particles were either arranged like beads in the central part of the fibers or formed clusters protruding from the fibers. In the PLGA-ND membranes, the fibers were thicker (diameter 270 ± 9 nm) than in pure PLGA meshes (diameter 218 ± 4 nm), but the areas of pores among these fibers were smaller than in pure PLGA samples (0.46 ± 0.02 µm² versus 1.28 ± 0.09 µm² in pure PLGA samples). The PLGA-ND membranes showed higher mechanical resistance, as demonstrated by rupture tests of load and deflection of rupture probe at failure. Both types of membranes enabled the attachment, spreading, and subsequent proliferation of human osteoblast-like MG-63 cells to a similar extent, although these values were usually lower than on polystyrene dishes. Nevertheless, the cells on both types of membranes were polygonal or spindle-like in shape, and were distributed homogeneously on the samples. From days 1–7 after seeding, their number rose continuously, and at the end of the experiment, these cells were able to create a confluent layer. At the same time, the cell viability, evaluated by a LIVE/DEAD viability/cytotoxicity kit, ranged from 92% to 97% on both types of membranes. In addition, on PLGA-ND membranes, the cells formed well developed talin-containing focal adhesion plaques. As estimated by the determination of tumor necrosis factor-alpha levels in the culture medium and concentration of intercellular adhesion molecule-1, MG-63 cells, and RAW 264.7 macrophages on these membranes did not show considerable inflammatory activity.
Conclusion: This study shows that nanofibrous PLGA membranes loaded with diamond nanoparticles have interesting potential for use in bone tissue engineering.

Keywords: nanofibers, nanoparticles, electrospinning, nanotechnology, regenerative medicine, human bone cells