Biological and physiochemical studies of electrospun polylactid/polyhydroxyoctanoate PLA/P(3HO) scaffolds for tissue engineering applications.

Polyhydroxyoctanoate, as a biocompatible and biodegradable biopolymer, represents an ideal candidate for biomedical applications. However, physical properties make it unsuitable for electrospinning, currently the most widely used technique for fabrication of fibrous scaffolds. To overcome this, it was blended with polylactic acid and polymer blend fibrous biomaterials were produced by electrospinning. The obtained PLA/PHO fibers were cylindrical, smaller in size, more hydrophilic and had a higher degree of biopolymer crystallinity and more favorable mechanical properties in comparison to the pure PLA sample. Cytotoxicity evaluation with human lung fibroblasts (MRC5 cells) combined with confocal microscopy were used to visualize mouse embryonic fibroblasts (MEF 3T3 cell line) migration and distribution showed that PLA/PHO samples support exceptional cell adhesion and viability, indicating excellent biocompatibility. The obtained results suggest that PLA/PHO fibrous biomaterials can be potentially used as biocompatible, biomimetic scaffolds for tissue engineering applications.

Vimentin Cytoskeleton Architecture Analysis on Polylactide and Polyhydroxyoctanoate Substrates for Cell Culturing.

Polylactide (PLA), widely used in bioengineering and medicine, gained popularity due to its biocompatibility and biodegradability. Natural origin and eco-friendly background encourage the search of novel materials with such features, such as polyhydroxyoctanoate (P(3HO)), a polyester of bacterial origin. Physicochemical features of both P(3HO) and PLA have an impact on cellular response 32, i.e., adhesion, migration, and cell morphology, based on the signaling and changes in the architecture of the three cytoskeletal networks: microfilaments (F-actin), microtubules, and intermediate filaments (IF). To investigate the role of IF in the cellular response to the substrate, we focused on vimentin intermediate filaments (VIFs), present in mouse embryonic fibroblast cells (MEF). VIFs maintain cell integrity and protect it from external mechanical stress, and also take part in the transmission of signals from the exterior of the cell to its inner organelles, which is under constant investigation. Physiochemical properties of a substrate have an impact on cells' morphology, and thus on cytoskeleton network signaling and assembly. In this work, we show how PLA and P(3HO) crystallinity and hydrophilicity influence VIFs, and we identify that two different types of vimentin cytoskeleton architecture: network "classic" and "nutshell-like" are expressed by MEFs in different numbers of cells depending on substrate features.

Physicochemical and Biological Characterisation of Diclofenac Oligomeric Poly(3-hydroxyoctanoate) Hybrids as ß-TCP Ceramics Modifiers for Bone Tissue Regeneration.

Nowadays, regenerative medicine faces a major challenge in providing new, functional materials that will meet the characteristics desired to replenish and grow new tissue. Therefore, this study presents new ceramic-polymer composites in which the matrix consists of tricalcium phosphates covered with blends containing a chemically bounded diclofenac with the biocompatible polymer-poly(3-hydroxyoctanoate), P(3HO). Modification of P(3HO) oligomers was confirmed by NMR, IR and XPS. Moreover, obtained oligomers and their blends were subjected to an in-depth characterisation using GPC, TGA, DSC and AFM. Furthermore, we demonstrate that the hydrophobicity and surface free energy values of blends decreased with the amount of diclofenac modified oligomers. Subsequently, the designed composites were used as a substrate for growth of the pre-osteoblast cell line (MC3T3-E1). An in vitro biocompatibility study showed that the composite with the lowest concentration of the proposed drug is within the range assumed to be non-toxic (viability above 70%). Cell proliferation was visualised using the SEM method, whereas the observation of cell penetration into the scaffold was carried out by confocal microscopy. Thus, it can be an ideal new functional bone tissue substitute, allowing not only the regeneration and restoration of the defect but also inhibiting the development of chronic inflammation.

Vimentin Association with Nuclear Grooves in Normal MEF 3T3 Cells.

Vimentin, an intermediate filament protein present in leukocytes, blood vessel endothelial cells, and multiple mesenchymal cells, such as mouse embryonic fibroblasts (MEF 3T3), is crucial for various cellular processes, as well as for maintaining the integrity and durability (stability) of the cell cytoskeleton. Vimentin intermediate filaments (VIFs) adhere tightly to the nucleus and spread to the lamellipodium and tail of the cell, serving as a connector between the nucleus, and the cell's edges, especially in terms of transferring mechanical signals throughout the cell. How these signals are transmitted exactly remains under investigation. In the presented work, we propose that vimentin is involved in that transition by influencing the shape of the nucleus through the formation of nuclear blebs and grooves, as demonstrated by microscopic observations of healthy MEF (3T3) cells. Grooved, or "coffee beans" nuclei, have, to date, been noticed in several healthy cells; however, these structures are especially frequent in cancer cells-they serve as a significant marker for recognition of multiple cancers. We observed 288 MEF3T3 cells cultured on polyhydroxyoctanoate (PHO), polylactide (PLA), and glass, and we identified grooves, coaligned with vimentin fibers in the nuclei of 47% of cells cultured on PHO, 50% of cells on glass, and 59% of cells growing on PLA. We also observed nuclear blebs and associated their occurrence with the type of substrate used for cell culture. We propose that the higher rate of blebs in the nuclei of cells, cultured on PLA, is related to the microenvironmental features of the substrate, pH in particular.

Insights into In Vitro Wound Closure on Two Biopolyesters-Polylactide and Polyhydroxyoctanoate.

Two bio-based polymers have been compared in this study, namely: polylactide (PLA) and polyhydroxyoctanoate (PHO). Due to their properties such as biocompatibility, and biointegrity they are considered to be valuable materials for medical purposes, i.e., creating scaffolds or wound dressings. Presented biopolymers were investigated for their impact on cellular migration strategies of mouse embryonic fibroblasts (MEF) 3T3 cell line. Advanced microscopic techniques, including confocal microscopy and immunofluorescent protocols, enabled the thorough analysis of the cell shape and migration. Application of wound healing assay combined with dedicated software allowed us to perform quantitative analysis of wound closure dynamics. The outcome of the experiments demonstrated that the wound closure dynamics for PLA differs from PHO. Single fibroblasts grown on PLA moved 1.5-fold faster, than those migrating on the PHO surface. However, when a layer of cells was considered, the wound closure was by 4.1 h faster for PHO material. The accomplished work confirms the potential of PLA and PHO as excellent candidates for medical applications, due to their properties that propagate cell migration, vitality, and proliferation-essential cell processes in the healing of damaged tissues.

Cellular architecture and migration behavior of fibroblast cells on polyhydroxyoctanoate (PHO): A natural polymer of bacterial origin.

Biodegradable and biocompatible novel materials of natural origin are gaining more and more attention in recent years. These so called biopolymers, characterized by their biointegrity and biocompatibility, find completely new and promising applications in biomedical sciences. The presented work focuses on the medium chain length elastomeric polyhydroxyalkanoate biopolymer-polyhydroxyoctanoate (PHO). This biopolymer is fully biodegradable without formation of harmful byproducts.We investigated PHO's physical properties with nanoindentation technique and scratch testing to determine Young's modulus and friction coefficient. Further, the work focused on the impact of PHO, used as growth substrate, on the physiology and morphology of mouse embryonic fibroblast cells (MEF 3T3). Application of fluorescent staining protocols and advanced microscopic techniques allowed to study the morphological changes in the cytoskeletons of cells grown on PHO and also gave an insight into their migration strategies on the polymer surface. We found that PHO exhibits no cellular cytotoxicity, similarly to a glass substrate. MEF cells spread better on glass surface than on each tested PHO substrate though there was almost no difference between PHO substrates cast from different solvents. However, a detailed analysis of actin and microtubule cytoskeletal architecture reveals changes in the density of actin and microtubular networks. Migration of MEF cells on PHO substrates was slower than on the glass substrate. To elucidate the molecular mechanisms of observed changes in cytoskeletal architecture and migration parameters can be of special interest for future medical application of PHO polymer.