Yury Rochev

Macromolecular Crowding Meets Tissue Engineering by Self‐Assembly: A Paradigm Shift in Regenerative Medicine

MMC, the addition of inert polydispersed macromolecules in the culture media, effectively emulates the dense in vivo extracellular space, resulting in amplified deposition of ECM in vitro and subsequent production of cohesive, ECM-rich living substitutes.

Long-term exposure of CdTe quantum dots on PC12 cellular activity and the determination of optimum non-toxic concentrations for biological use.

BackgroundThe unique and tuneable photonic properties of Quantum Dots (QDs) have made them potentially useful tools for imaging biological entities. However, QDs though attractive diagnostic and therapeutic tools, have a major disadvantage due to their inherent cytotoxic nature. The cellular interaction, uptake and resultant toxic influence of CdTe QDs (gelatinised and non-gelatinised Thioglycolic acid (TGA) capped) have been investigated with pheochromocytoma 12 (PC12) cells. In conjunction to their analysis by confocal microscopy, the QD - cell interplay was explored as the QD concentrations were varied over extended (up to 72 hours) co-incubation times. Coupled to this investigation, cell viability, DNA quantification and cell proliferation assays were also performed to compare and contrast the various factors leading to cell stress and ultimately death.ResultsThioglycolic acid (TGA) stabilised CdTe QDs (gel and non - gel) were co-incubated with PC12 cells and investigated as to how their presence influenced cell behaviour and function. Cell morphology was analysed as the QD concentrations were varied over co-incubations up to 72 hours. The QDs were found to be excellent fluorophores, illuminating the cytoplasm of the cells and no deleterious effects were witnessed at concentrations of ~10-9 M. Three assays were utilised to probe how individual cell functions (viability, DNA quantification and proliferation) were affected by the presence of the QDs at various concentrations and incubation times. Cell response was found to not only be concentration dependant but also influenced by the surface environment of the QDs. Gelatine capping on the surface acts as a barrier towards the leaking of toxic atoms, thus reducing the negative impact of the QDs.ConclusionThis study has shown that under the correct conditions, QDs can be routinely used for the imaging of PC12 cells with minimal adverse effects. We have found that PC12 cells are highly susceptible to an increased concentration range of the QDs, while the gelatine coating acts as a barrier towards enhanced toxicity at higher QD concentrations.

Straightforward, One-Step Fabrication of Ultrathin Thermoresponsive Films from Commercially Available pNIPAm for Cell Culture and Recovery

The use of thermoresponsive surfaces as platforms for cell culture and cell regeneration has been explored over the last couple of decades. Poly-N-isopropylacrylamide (pNIPAm) is a well characterized thermoresponsive polymer which has an aqueous lower critical solution temperature (LCST) in a physiologically useful range, which allows it to reversibly attract (T < 32 °C) and repel water (T > 32 °C). It is this phenomenon that is exploited in temperature-controlled cell harvesting. pNIPAm coatings are generally poorly cell compatible and a number of complex or expensive techniques have been developed in order to overcome this issue. This study seeks to design a simple one-step system whereby commercially sourced pNIPAm is used to achieve similar results. Films were deposited using the operationally simple but rheologically complex spin coating technique. Reversible temperature modulated cell adhesion was achieved using a variety of different cell lines. This system offers a simplistic and cheaper alternative to methods used elsewhere.

Ultra-thin spin coated crosslinkable hydrogels for use in cell sheet recovery—synthesis, characterisation to application

The advantages of detaching adherent cells from thermoresponsive platforms over conventional cell detachment protocols has been well documented. This study focuses on the development of an alternative method to produce thermoresponsive surfaces for cell and cell sheet regeneration to already established techniques which are complex and expensive and may be inaccessible to many laboratories. A photcrosslinkable poly-N-isopropylacrylamide (pNIPAm) copolymer was synthesised and thin films of the copolymer were deposited using the operationally simple spin coating technique which were subsequently crosslinked upon exposure to ultraviolet (UV) irradiation. Characterisation of hydrogel properties and behaviour was achieved using UV spectroscopy, atomic force microscopy (AFM), advancing contact angle, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR) and white light interferometry analyses. Results indicate that 3T3 fibroblast cells adhere and proliferate to confluence on hydrogels of 30 nm was comparatively poor. Confluent cell sheets were harvested from the sub 30 nm ultra-thin hydrogels upon temperature reduction within 10 min. Spin coating allows for the facile control of film thickness via variation of the depositing polymer solution concentration and therefore the routine fabrication of ultra-thin hydrogels capable of hosting cells to confluence is easily achievable.

Cell and cell sheet recovery from pNIPAm coatings; motivation and history to present day approaches

The regeneration of cells and cell sheets mediated by thermoresponsive substrates represents an important and ever growing area in tissue engineering. This review seeks to track the development of this field from inception to the present day by highlighting the most significant breakthroughs as well as focusing on important physical and chemical characterization of substrates produced for this specific purpose. Furthermore, a critical evaluation encompassing the advantages and disadvantages of different techniques used for producing such surfaces will be included as well as suggestions for possible future directions.

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

Surface modification for controlled cell growth on copolymers of N -isopropylacrylamide

We investigated the behaviour of cell cultures on the surface of thermoresponsive polymers. We synthesised a series of copolymers of N-isopropylacrylamide and N-tert -butylacrylamide (NtBA) with ratios of 85:15, 65:35 and 50:50. Increasing the amount of NtBA results in a reduction in the lower critical solution temperature as determined by microcalorimetric methods and an increase in surface hydrophobicity. Experiments determined that human epithelial cell growth was almost identical on surfaces with a higher degree of hydrophobicity. Preconditioning of surfaces with cell culture medium containing serum promoted cell adhesion on more hydrophilic polymers.

Nanomechanical properties of poly(lactic-co-glycolic) acid film during degradation

Despite the potential applications of poly(lactic-co-glycolic) acid (PLGA) coatings in medical devices, the mechanical properties of this material during degradation are poorly understood. In the present work, the nanomechanical properties and degradation of PLGA film were investigated. Hydrolysis of solvent-cast PLGA film was studied in buffer solution at 37 °C. The mass loss, water uptake, molecular weight, crystallinity and surface morphology of the film were tracked during degradation over 20 days. Characterization of the surface hardness and Youngs modulus was performed using the nanoindentation technique for different indentation loads. The initially amorphous films were found to remain amorphous during degradation. The molecular weight of the film decreased quickly during the initial days of degradation. Diffusion of water into the film resulted in a reduction in surface hardness during the first few days, followed by an increase that was due to the surface roughness. There was a significant delay between the decrease in the mechanical properties of the film and the decrease in the molecular weight. A sudden decline in mechanical properties indicated that significant bulk degradation had occurred.

An Investigation into the Effect of Surface Roughness of Stainless Steel on Human Umbilical Vein Endothelial Cell Gene Expression

The surface properties of vascular devices dictate the initial postimplantation reactions that occur and thus the efficacy of the implantation procedure. Over the last number of years, a number of different stent designs have emerged and stents are generally polished to a mirror finish during the manufacturing procedure. This study sought to investigate the effect of stainless steel surface roughness on endothelial cell gene expression using an appropriate cell culture in vitro assay system. Stainless steel discs were roughened by shot blasting or polished by mechanical polishing. The surface roughness of the treated and untreated discs was determined by atomic force microscopy (AFM). Cells were seeded on collagen type 1 gels and left to attach for 24 h. Stainless steel discs of varying roughness were then placed in contact with the cells and incubated for 24 h. RNA extractions and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was then performed to determine the expression levels of candidate genes in the treated cells compared to suitable control cells. E-selectin and vascular cellular adhesion molecule (VCAM-1) were found to be significantly up-regulated in cells incubated with polished and roughened samples, indicating endothelial cell activation and inflammation. This study indicates that the surface roughness of stainless steel is an important surface property in the development of vascular stents.

Effects of long-term exposure of gelatinated and non-gelatinated cadmium telluride quantum dots on differentiated PC12 cells

BackgroundThe inherent toxicity of unmodified Quantum Dots (QDs) is a major hindrance to their use in biological applications. To make them more potent as neuroprosthetic and neurotherapeutic agents, thioglycolic acid (TGA) capped CdTe QDs, were coated with a gelatine layer and investigated in this study with differentiated pheochromocytoma 12 (PC12) cells. The QD - cell interactions were investigated after incubation periods of up to 17 days by MTT and APOTOX-Glo Triplex assays along with using confocal microscopy.ResultsLong term exposure (up to 17 days) to gelatinated TGA-capped CdTe QDs of PC12 cells in the course of differentiation and after neurites were grown resulted in dramatically reduced cytotoxicity compared to non-gelatinated TGA-capped CdTe QDs.ConclusionThe toxicity mechanism of QDs was identified as caspase-mediated apoptosis as a result of cadmium leaking from the core of QDs. It was therefore concluded that the gelatine capping on the surface of QDs acts as a barrier towards the leaking of toxic ions from the core QDs in the long term (up to 17 days).