The Effect of Osteoblast Isolation Methods from Adult Rats on Osteoclastogenesis in Co-Cultures Radmila Žižková, Věra Hedvičáková, Veronika Hefka Blahnová, Věra Sovková, Michala Rampichová, et al. International Journal of Molecular Sciences, 2022 Co-cultures of osteoblasts and osteoclasts are on the rise because they enable a more complex study. Diseases such as osteoporosis are related to a higher age. Thus, cell isolation from adult individuals is necessary. Osteoblasts can be isolated from the rat femur by three methods: explant culture, explant culture with enzymatic pre-treatment, or enzymatic treatment. The isolation methods yield different populations of osteoblasts which, in a co-culture with peripheral blood mononuclear cells, might result in differences in osteoclastogenesis. Therefore, we examined the differences in osteogenic markers, cell proliferation, and the metabolic activity of isolated osteoblast-like cells in a growth and differentiation medium. We then evaluated the effect of the isolated populations of osteoblast-like cells on osteoclastogenesis in a subsequent co-culture by evaluating osteoclast markers, counting formed osteoclast-like cells, and analyzing their area and number of nuclei. Co-cultures were performed in the presence or absence of osteoclastogenic growth factors, M-CSF and RANKL. It was discovered that enzymatic isolation is not feasible in adult rats, but explant culture and explant culture with enzymatic pre-treatment were both successful. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation than explant culture in a growth medium. The differentiation medium reduced differences in proliferation during the culture. Some differences in metabolic activity and ALP activity were also found between the osteoblast-like cells isolated by explant culture or by explant culture with enzymatic pre-treatment, but only on some days of cultivation. According to microscopy, the presence of exogenous growth factors supporting osteoclastogenesis in co-cultures was necessary for the formation of osteoclast-like cells. In this case, the formation of a higher number of osteoclast-like cells with a larger area was observed in the co-culture with osteoblast-like cells isolated by explant culture compared to the explant culture with enzymatic pre-treatment. Apart from this observation, no differences in osteoclast markers were noted between the co-cultures with osteoblast-like cells isolated by explant culture and the explant culture with enzymatic pre-treatment. The TRAP and CA II activity was higher in the co-cultures with exogenous growth than that in the co-cultures without exogenous growth factors on day 7, but the opposite was true on day 14. To conclude, explant culture and explant culture with enzymatic pre-treatment are both suitable methods to yield osteoblast-like cells from adult rats capable of promoting osteoclastogenesis in a direct co-culture with peripheral blood mononuclear cells. Explant culture with enzymatic pre-treatment yielded cells with a higher proliferation. The explant culture yielded osteoblast-like cells which induced the formation of a higher number of osteoclast-like cells with a larger area compared to the explant culture with enzymatic pre-treatment when cultured with exogenous M-CSF and RANKL.
Cellular response to individual components of the platelet concentrate Vera Sovkova, Karolina Vocetkova, Věra Hedvičáková, Veronika Hefka Blahnová, Matěj Buzgo, et al. International Journal of Molecular Sciences, 2021 Platelet concentrates and especially their further product platelet lysate, are widely used as a replacement for cell culturing. Platelets contain a broad spectrum of growth factors and bioactive molecules that affect cellular fate. However, the cellular response to individual components of the human platelet concentrate is still unclear. The aim of this study was to observe cellular behavior according to the individual components of platelet concentrates. The bioactive molecule content was determined. The cells were supplemented with a medium containing 8% (v/v) of platelet proteins in plasma, pure platelet proteins in deionized water, and pure plasma. The results showed a higher concentration of fibrinogen, albumin, insulin growth factor I (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), in the groups containing plasma. On the other hand, chemokine RANTES and platelet-derived growth factor bb (PDGF-bb), were higher in the groups containing platelet proteins. The groups containing both plasma and plasma proteins showed the most pronounced proliferation and viability of mesenchymal stem cells and fibroblasts. The platelet proteins alone were not sufficient to provide optimal cell growth and viability. A synergic effect of platelet proteins and plasma was observed. The data indicated the importance of plasma in platelet lysate for cell growth.
Fdm 3d printed composites for bone tissue engineering based on plasticized poly(3-hydroxybutyrate)/poly(d,l-lactide) blends Veronika Melčová, Kateřina Svoradová, Přemysl Menčík, Soňa Kontárová, Michala Rampichová, et al. Polymers, 2020 Tissue engineering is a current trend in the regenerative medicine putting pressure on scientists to develop highly functional materials and methods for scaffolds’ preparation. In this paper, the calibrated filaments for Fused Deposition Modeling (FDM) based on plasticized poly(3-hydroxybutyrate)/poly(d,l-lactide) 70/30 blend modified with tricalcium phosphate bioceramics were prepared. Two different plasticizers, Citroflex (n-Butyryl tri-n-hexyl citrate) and Syncroflex (oligomeric adipate ester), both used in the amount of 12 wt%, were compared. The printing parameters for these materials were optimized and the printability was evaluated by recently published warping test. The samples were studied with respect to their thermal and mechanical properties, followed by biological in vitro tests including proliferation, viability, and osteogenic differentiation of human mesenchymal stem cells. According to the results from differential scanning calorimetry and tensile measurements, the Citroflex-based plasticizer showed very good softening effect at the expense of worse printability and unsatisfactory performance during biological testing. On the other hand, the samples with Syncroflex demonstrated lower warping tendency compared to commercial polylactide filament with the warping coefficient one third lower. Moreover, the Syncroflex-based samples exhibited the non-cytotoxicity and promising biocompatibility.
A simple drug delivery system for platelet-derived bioactive molecules, to improve melanocyte stimulation in vitiligo treatment Karolina Vocetkova, Vera Sovkova, Matej Buzgo, Vera Lukasova, Radek Divin, et al. Nanomaterials, 2020 Vitiligo is the most common depigmentation disorder of the skin. Currently, its therapy focuses on the halting of the immune response and stimulation of the regenerative processes, leading to the restoration of normal melanocyte function. Platelet-rich plasma (PRP) represents a safe and cheap regenerative therapy option, as it delivers a wide spectrum of native growth factors, cytokines and other bioactive molecules. The aim of this study was to develop a simple delivery system to prolong the effects of the bioactive molecules released from platelets. The surface of electrospun and centrifugally spun poly-ε-caprolactone (PCL) fibrous scaffolds was functionalized with various concentrations of platelets; the influence of the morphology of the scaffolds and the concentration of the released platelet-derived bioactive molecules on melanocytes, was then assessed. An almost two-fold increase in the amount of the released bioactive molecules was detected on the centrifugally spun vs. electrospun scaffolds, and a sustained 14-day release of the bioactive molecules was demonstrated. A strong concentration-dependent response of melanocyte to the bioactive molecules was observed; higher concentrations of bioactive molecules resulted in improved metabolic activity and proliferation of melanocytes. This simple system improves melanocyte viability, offers on-site preparation and is suitable for prolonged topical PRP administration.
In vitro evaluation of a novel nanostructured Ti-36Nb-6Ta alloy for orthopedic applications Barbora Voltrova, Petra Jarolimova, Vojtech Hybasek, Veronika Hefka Blahnova, Josef Sepitka, et al. Nanomedicine, 2020 Aim: To evaluate the impact of a nanostructured surface created on β-titanium alloy, Ti-36Nb-6Ta, on the growth and differentiation of human mesenchymal stem cells. Materials & methods: The nanotubes, with average diameters 18, 36 and 46 nm, were prepared by anodic oxidation. Morphology, hydrophilicity and mechanical properties of the nanotube layers were characterized. The biocompatibility and osteogenic potential of the nanostructured surfaces were established using various in vitro assays, scanning electron microscopy and confocal microscopy. Results: The nanotubes lowered elastic modulus close to that of bone, positively influenced cell adhesion, improved ALP activity, synthesis of type I collagen and osteocalcin expression, but diminished early cell proliferation. Conclusion: Nanostructured Ti-36Nb-6Ta with nanotube diameters 36 nm was the most promising material for bone implantation.
Testing of fibrous membranes with different scaffold morphology for development of 3D skin model Vera Sovková, Karolína Vocetková, Michala Rampichová, Eva Filová Key Engineering Materials, 2020 Replacement of in vivo testing using advanced 3D constructs is an important challenge in tissue engineering applications. The cell culture material should be biocompatible and should mimic the natural microenvironment of the existing tissue. Nanofibrous scaffolds prepared by electrospinning from biocompatible polymers have suitable properties for cell culture in a 3D environment. Thanks to the high volume-to-surface ratio, controlled porous structure with high pore interconnection and microarchitecture in the nanoscale range, nanofibers are in the foreground of interest. We tested membranes with different topography with keratinocyte and fibroblast cell lines. Fibroblast showed stable growth with no difference among the scaffolds. On the other hand, keratinocytes preferred scaffolds with nanofiber morphology.
Functionalization of polymeric nanofibers using platelets for melanocyte culture Karolina Vocetkova, Vera Sovkova, Matej Buzgo, Radek Divin, Evzen Amler, et al. Lekar A Technika, 2020 Tissue engineering is an interdisciplinary field that uses a combination of cells, suitable biomaterials and bioactive molecules to engineer the desired tissue and restore lost function. These principles have quickly begun to spread to the therapy of multiple diseases, including depigmentation disorders. The most common depigmentation disorder is vitiligo, a disease with deep psychosocial implications. Thanks to their unique properties, electrospun polymeric nanofibers represent a material suitable for tissue engineering applications. Furthermore, they may be functionalized with platelets, cells that contain a wide spectrum of growth factors and chemokines. The aim of this paper was to evaluate the functionalization of polymeric nanofibers with platelets and their effects in melanocyte culture. The scaffolds were visualized using scanning electron microscopy, the metabolic activity and proliferation of melanocytes was determined using MTS assay and dsDNA quantification, respectively. Furthermore, the melanocytes were stained and visualized using confocal microscopy. The acquired data showed that poly-ε-caprolactone functionalized with platelets promoted the viability and proliferation of melanocytes. According to the results, such a functionalized scaffold combining nanofibers and platelets may be suitable for melanocyte culture.
Coaxial nanofibrous scaffold prepared using centrifugal spinning as a drug delivery system for skeletal tissue engineering Michala Rampichová, Vera Lukášová, Matej Buzgo, Karolína Vocetková, Vera Sovková, et al. Key Engineering Materials, 2020 Skeletal disorders, caused by trauma, disease, or carcinoma, may result in tissue loss and, finally, in endoprosthesis. Tissue engineering offers an alternative - tissue scaffolds. Its constructs may be seeded with autologous cells or, alternatively, attract cells from the surrounding tissues. Such a scaffold must meet several requirements, such as biocompatibility, biodegradability and suitable morphology for cell attachment and proliferation. Nonetheless, scaffold should stimulate cells migrated from the surrounding tissues to infiltrate the scaffold, proliferate and differentiate to the required cell type. In the current study, we developed a fibrous scaffold with 3D structure using emulsion centrifugal spinning. The scaffold from poly-ɛ-caprolactone contained a cocktail of growth factors, i.e. TGF-β, IGF and bFGF. The released growth factors enhanced cell proliferation and chondrogenic differentiation. The scaffold is a promising material for skeletal tissue engineering.
The effect of poly-ϵ-caprolactone nanofibers with platelets on cell proliferation and viability Nanocon 2018 Conference Proceedings 10th Anniversary International Conference on Nanomaterials Research and Application, 2019
