Polychromatic light-induced osteogenic activity in 2D and 3D cultures.

Photobiomodulation (PBM) has been applied to manipulate cellular responses by using monochromatic light in different wavelengths from ultraviolet (UV) to infrared (IR) region. Until now, an effective wavelength has not been revealed to induce proliferation and/or differentiation of cells. Therefore, in the presented study, we decided to use a specially designed plasma arc light source providing wavelengths between 590 and 1500 nm in order to investigate its biomodulatory effects on chitosan scaffold-supported three-dimensional (3D) cell cultures. For comparison, two-dimensional (2D) cell cultures were also carried out in tissue-culture polystyrene dishes (TCPS). The results showed that light-induced temperature rise did not affect cells when the distance between the light source and the cells was 10 cm and the frequency of administration was daily. Moreover, light was applied for 5 and 10 min to the cells in TCPS and in chitosan scaffold groups, respectively. Cell culture studies under static conditions indicated that polychromatic light significantly stimulated bone nodule formation via the prolonged cell survival and stimulated differentiation of MC3T3-E1 preosteoblastic cells in both TCPS and chitosan scaffold groups. In conclusion, specially designed plasma arc light source used in this study induces formation of bone tissue and so, this light source is proposed as an appropriate system for in vitro bone tissue engineering applications. Statistical analyses were performed with one-way ANOVA by using GraphPad Instat software and standard deviations were calculated by using data of three parallel samples for each group.

A Silk Fibroin and Peptide Amphiphile-Based Co-Culture Model for Osteochondral Tissue Engineering.

New biomaterials with the properties of both bone and cartilage extracellular matrices (ECM) should be designed and used with co-culture systems to address clinically applicable osteochondral constructs. Herein, a co-culture model is described based on a trilayered silk fibroin-peptide amphiphile (PA) scaffold cultured with human articular chondrocytes (hACs) and human bone marrow mesenchymal stem cells (hBMSCs) in an osteochondral cocktail medium for the cartilage and bone sides, respectively. The presence of hACs in the co-cultures significantly increases the osteogenic differentiation potential of hBMSCs based on ALP activity, RT-PCR for osteogenic markers, calcium analyses, and histological stainings, whereas hACs produces a significant amount of glycosaminoglycans (GAGs) for the cartilage region, even in the absence of growth factor TGF-ß family in the co-culture medium. This trilayered scaffold with trophic effects offers a promising strategy for the study of osteochondral defects.

Synergistic effect of exogeneous and endogeneous electrostimulation on osteogenic differentiation of human mesenchymal stem cells seeded on silk scaffolds.

Bioelectrical regulation of bone fracture healing is important for many cellular events such as proliferation, migration, and differentiation. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk scaffolds in response to different modes of electrostimulation (e.g., exogeneous and/or endogeneous). Endogeneous electrophysiology was altered through the use of monensin (10 nM) and glibenclamide (10 µM), along with external electrostimulation (60 kHz; 100-500 mV). Monensin enhanced the expression of early osteogenic markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX-2). When exogeneous electrostimulation was combined with glibenclamide, more mature osteogenic marker upregulation based on bone sialoprotein expression (BSP) and mineralization was found. These results suggest the potential to exploit both exogeneous and endogeneous biophysical control of cell functions towards tissue-specific goals.

Double-effective chitosan scaffold-PLGA nanoparticle system for brain tumour therapy: in vitro study.

The chitosan scaffold, which has both of anticancer and antivascularization effects, was developed for using in local therapy of brain tumours. This is why, poly-lactic-co-glycolic acid (50:50) nanoparticles (~200 nm) including an anticancer drug, 5-fluorouracil (5-FU), were prepared by emulsion-solvent evaporation method. Then, these nanoparticles and antivascularization agent, bevacizumab, were loaded into the scaffold during manufacturing by freeze-drying and embedding after freeze-drying, respectively. The idea behind this system is to destroy tumour tissue by releasing 5-FU and to prevent the proliferation of tumour cells by releasing bevacizumab. In addition, 3D scaffold can support healthy tissue formation in the tumourigenic region. In vitro effectiveness of this system was investigated on T98G human glioblastoma cell line and human umbilical vein endothelial cells. The results show that the chitosan scaffold containing 100 µg 5-FU and 100 µg bevacizumab has a potential to prevent the tumour formation in vitro conditions.

Thermosensitive PHEMA microcarriers: ATRP synthesis, characterization, and usabilities in cell cultures.

In this study, we developed a novel microcarrier to enhance the production of anchorage-dependent mammalian cells in large scale by preserving them from the effects of shear forces and to enhance their removal from the surface without using proteolytic enzymes and chelating agents. This 'thermosensitive microcarrier' was synthesized by the grafting thermoresponsive molecule, N-isopropylacrylamide (NIPAAm), to the crosslinked poly(2-hydroxyethyl methacrylate) (PHEMA) beads by surface-initiated atom transfer radical polymerization. NIPAAm was polymerized on bromine-activated beads' surfaces to prepare PHEMA-g-PNIPAAm microcarriers. Then, they were chemically characterized by attenuated total reflectance Fourier transform infrared and electron spectroscopy for chemical analysis. Surface morphologies were further investigated by scanning electron microscope and atomic force microscopy techniques. The results of characterization studies confirmed that PNIPAAm was successfully grafted onto PHEMA beads by the means of atom transfer radical polymerization reaction. The cellular activities of PHEMA-g-PNIPAAm microcarriers were evaluated at static and dynamic culture conditions by using two types of cell lines with different morphology, i.e. L929 mouse fibroblasts and HS2 epithelial human keratinocytes. The microcarriers exhibited better cell adhesion and proliferation characteristics for both cell lines. Although their thermally induced cell detachment efficiencies are lower than that of trypsinization, thermally harvested cells preserved their surface morphologies and proliferation characteristics.

PNIPAAm-grafted thermoresponsive microcarriers: surface-initiated ATRP synthesis and characterization.

In this study, we developed novel thermoresponsive microcarriers as a powerful tool for cell culture and tissue engineering applications. For this purpose, two types of commercially available spherical microparticles (approximately 100 µm in diameter), dextran-based Sephadex® and vinyl acetate-based VA-OH (Biosynth®), were used and themoresponsive poly(N-isopropylacrylamide) (PNIPAAm) was grafted to the beads' surfaces by surface-initiated atom transfer radical polymerization (SI-ATRP). Initially, hydroxyl groups of microbeads were reacted with 2-bromopropionyl bromide to form ATRP macroinitiator. Then, NIPAAm was successfully polymerized from the initiator attached microbeads by ATRP with CuBr/2,2'-dipyridyl, catalyst complex. Furthermore, grafted and ungrafted microbeads were characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscope (SEM), atomic force microscopy (AFM) and electron spectroscopy for chemical analysis (ESCA). The results of characterization studies confirmed that PNIPAAm was successfully grafted onto both dextran and vinyl acetate-based beads by means of ATRP reaction and thus, grafted microbeads gained thermoresponsive characteristics which will be evaluated for cell harvesting in further studies.