Guided tissue regeneration (GTR) is a surgical procedure that specifically aims to regenerate soft and hard periodontal tissues when they are irreversibly destructed. GTR requires special porous barrier membranes, preferentially degradable, capable of preventing soft tissue infiltration into the bone defect and simultaneously supporting bone regeneration. The aim of this study was to produce a degradable poly(L-lactide-co-glycolide) (PLGA) membrane with appropriate microstructure, i.e. less porous on the surface intended to be in contact with the gum, while being more porous on the surface contacting the bone tissue defect to promote osteogenic cell adhesion, proliferation, and differentiation, and thus bone tissue restoration. In addition to microstructural cues, we provide our membranes with arginine-glycine-aspartic acid (RGD) motifs, which may act as biochemical cues supporting specific integrin-mediated cell adhesion.
The membranes were produced using poly(L-lactide-co-glycolide) (PLGA, 85:15, Mn = 100 kDa, d = 1.9). Poly(2-metyl-2-oxazoline-b-2-butyl-2-oxazoline-b-2-methyl-2-oxazoline) (POx), was modified with an RGD derivative with 6-aminohexanoic acid (POx-RGD). To obtain the membranes, we co-dissolved PLGA, PEG, and POx_RGD in DCM, solvent-casted, dried, followed by PEG leaching . Raman spectroscopy, FTIR-ATR, and XPS were used to characterise all ingredients and the membranes. The membranes were also characterized using SEM; mechanical properties, susceptibility to degradation, wettability, and surface free energy were also assessed. Osteoblast-like MG-63 cells were cultured for 4, 24 and 96 h on the membranes and analyzed by metabolic activity and live/dead tests, as well as by phalloidin/DAPI fluorescent staining to visualize cell morphology and cytoskeleton reorganization.
PLGA, PEG, POx, RGD, and POx_RGD were characterized using Raman, FTIR-ATR, and XPS spectroscopic techniques. Detailed analysis of the spectra confirmed that RGD was successfully coupled with POx. The membranes for GTR were obtained by phase separation and preferential adsorption of POx_RGD molecules at the PLGA/PEG interface with POx_RGD exposed to hydrophilic PEG, followed by solvent evaporation and PEG leaching. The membranes had an asymmetric microstructure, as shown in the SEM pictures of both the surfaces and cross-sections; the glass-cured surface was more porous and was characterized by a higher surface area as compared to the air-cured surface. XPS and FTIR-ATR studies confirmed that POx_RGD was immobilized on the membrane surface; however, this modification practically did not influence the surface wettability and surface free energy values. In vitro tests showed that the POx_RGD-modified PLGA membranes supported osteoblast-like cell adhesion, proliferation, and viability to the highest extent, compared to membranes without modification or modified only with POx.
The one-step phase separation process between PLGA, PEG, and POx_RGD dissolved in DCM, followed by drying and leaching of PEG, resulted in asymmetric PLGA membranes with enhanced biological properties, which could be considered for the guided tissue regeneration technique in periodontology and bone tissue engineering.
This study was supported by the program ‘Excellence Initiative-Research University’ and from the subsidy of the Ministry of Education and Science for the AGH University of Science and Technology in Kraków, Poland (Project No 220.127.116.117).
 Tryba, A.M. et al., J. Funct. Biomater. 2022, 13, 4"