176 research outputs found
Poly(n-butyl Methacrylate) with Primary Amine End Groups for Supporting Cell Adhesion and Proliferation of Renal Epithelial Cells
Polymer coatings that support epithelial cell culture have been developed. Ozonolysis and subsequent work up of poly(butyl methacrylate-co-butadiene) copolymers is used to form oligomers with carboxylic acid end groups, which are then further reacted with diamines to provide poly(butyl methacrylate)s with primary amine end groups. The polymers are cast as films and used as cell culture substrates for human dermal fibroblasts and human renal epithelial cells. Fibroblast and epithelial cells adhere and proliferate on acid functional materials but on amine functional films epithelial cells show greater viability than fibroblasts
A new mode of contrast in biological second harmonic generation microscopy
Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature
Porous microspheres support mesenchymal progenitor cell ingrowth and stimulate angiogenesis
Porous microspheres have the potential for use as injectable bone fillers to obviate
the need for open surgery. Successful bone fillers must be able to support
vascularisation since tissue engineering scaffolds often cease functioning soon after
implantation due to a failure to vascularise rapidly. Here, we test the angiogenic
potential of a tissue engineered bone filler based on a photocurable acrylate-based
high internal phase emulsion (HIPE). Highly porous microspheres were fabricated
via two processes, which were compared. One was taken forward and investigated
for its ability to support human mesenchymal progenitor cells and angiogenesis in a
chorioallantoic membrane (CAM) assay. Porous microspheres with either a narrow
or broad size distribution were prepared via a T-junction microfluidic device or by a
controlled stirred-tank reactor of the HIPE water in oil in water (w/o/w), respectively.
Culture of human embryonic stem cell-derived mesenchymal progenitor (hES-MP)
cells showed proliferation over 11 days and formation of cell-microsphere aggregates.
In-vitro, hES-MP cells were found to migrate into microspheres through their
surface pores over time. The presence of osteoblasts, differentiated from the hES-MP
cells, was evidenced through the presence of collagen and calcium after 30 days.
Microspheres pre-cultured with cells were implanted into CAM for 7 days and compared
with control microspheres without pre-cultured cells. The hES-MP seeded
microspheres supported greater angiogenesis, as measured by the number of blood
vessels and bifurcations, while the empty scaffolds attracted host chick cell ingrowth.
This investigation shows that controlled fabrication of porous microspheres has the
potential to create an angiogenic, bone filling material for use as a cell delivery
vehicle
Elastomeric, bioadhesive and pH-responsive amphiphilic copolymers based on direct crosslinking of poly(glycerol sebacate)-co-polyethylene glycol
Poly(glycerol sebacate) (PGS), a synthetic biorubber, is characterised by its biocompatibility, high elasticity and tunable mechanical properties; however, its inherent hydrophobicity and insolubility in water make it unsuitable for use in advanced biomaterials like hydrogels fabrication. Here, we developed new hydrophilic PGS-based copolymers that enable hydrogel formation through use of two different types of polyethylene glycol (PEG), polyethylene glycol (PEG2) or glycerol ethoxylate (PEG3), combined at different ratios. A two-step polycondensation reaction was used to produce poly(glycerol sebacate)-co-polyethylene glycol (PGS-co-PEG) copolymers that were then crosslinked thermally without the use of initiators or crosslinkers, resulting in PGS-co-PEG2 and PGS-co-PEG3 amphiphilic polymers. It has been illustrated that the properties of PGS-co-PEG copolymers can be controlled by altering the type and amount of PEG. PGS-co-PEG copolymers containing PEG ≥ 40% showed high swelling, flexibility, stretching, bioadhesion and biocompatibility, and good enzymatic degradation and mechanical properties. Also, the addition of PEG created hydrogels that demonstrated pH-responsive behaviours, which can be used for bioapplications requiring responding to physicochemical dynamics. Interestingly, PGS-co-40PEG2 and PGS-co-60PEG3 had the highest shear strengths, 340.4 ± 49.7 kPa and 336.0 ± 35.1 kPa, and these are within the range of commercially available sealants or bioglues. Due to the versatile multifunctionalities of these new copolymer hydrogels, they can have great potential in soft tissue engineering and biomedicine
The importance of mimicking dermal-epidermal junction for skin tissue engineering : a review
There is a distinct boundary between the dermis and epidermis in the human skin called the basement membrane, a dense collagen network that creates undulations of the dermal–epidermal junction (DEJ). The DEJ plays multiple roles in skin homeostasis and function, namely, enhancing the adhesion and physical interlock of the layers, creating niches for epidermal stem cells, regulating the cellular microenvironment, and providing a physical boundary layer between fibroblasts and keratinocytes. However, the primary role of the DEJ has been determined as skin integrity; there are still aspects of it that are poorly investigated. Tissue engineering (TE) has evolved promising skin regeneration strategies and already developed TE scaffolds for clinical use. However, the currently available skin TE equivalents neglect to replicate the DEJ anatomical structures. The emergent ability to produce increasingly complex scaffolds for skin TE will enable the development of closer physical and physiological mimics to natural skin; it also allows researchers to study the DEJ effect on cell function. Few studies have created patterned substrates that could mimic the human DEJ to explore their significance. Here, we first review the DEJ roles and then critically discuss the TE strategies to create the DEJ undulating structure and their effects. New approaches in this field could be instrumental for improving bioengineered skin substitutes, creating 3D engineered skin, identifying pathological mechanisms, and producing and screening drugs
Oxygen mapping of melanoma spheroids using small molecule platinum probe and phosphorescence lifetime imaging microscopy
Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application
In vitro low-fluence photodynamic therapy parameter screening using 3D tumor spheroids shows that fractionated light treatments enhance phototoxicity
The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to the limitations of two-dimensional cell culture and multiple parameters (dose, light intensity, uptake time), which complicate progression to in vivo experiments and clinical translation. Three-dimensional (3D) cell culture models like multicellular cancer tumor spheroids (MCTS) show great similarities to in vivo avascular tumor conditions, improving the speed and accuracy of screening novel compounds with various treatment combinations. In this study, we utilize C8161 human melanoma spheroids to screen PDT treatment combinations using protoporphyrin IX (PpIX) and drug-loaded carbon dot (CD) conjugates PpIX-CD and PpIX@CD at ultralow fluence values (<10 J/cm2). Conjugates show equivalent light-induced damage to PpIX from 1 μg/mL with significantly less dark cytotoxicity up to 72 h after exposure, shown by LDH release and dsDNA content. Fractionated treatments, carried out by dividing light exposure with 24 h intervals, demonstrate an enhanced PDT effect compared to single exposure at equal concentrations. Light sheet fluorescence microscopy combined with live/dead staining demonstrates that spheroids sustain extensive damage after PDT, with PpIX and PpIX-CD showing improved uptake compared to PpIX@CD. We show that PDT parameter screening can be carried out using a low-cost and convenient combination of assays to improve the efficiency of evaluating novel compounds
Inhibition and reversal of a TGF-β1 induced myofibroblast phenotype by adipose tissue-derived paracrine factors
Background
Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation.
Methods
The transforming growth factor-β1 (TGF-β1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts.
Results
Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-β1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue.
Conclusions
Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism
Development of PCL polyHIPE substrates for 3D breast cancer cell culture
Cancer is a becoming a huge social and economic burden on society, becoming one of the most significant barriers to life expectancy in the 21st century. In particular, breast cancer is one of the leading causes of death for women. One of the most significant difficulties to finding efficient therapies for specific cancers, such as breast cancer, is the efficiency and ease of drug development and testing. Tissue-engineered (TE) in vitro models are rapidly developing as an alternative to animal testing for pharmaceuticals. Additionally, porosity included within these structures overcomes the diffusional mass transfer limit whilst enabling cell infiltration and integration with surrounding tissue. Within this study, we investigated the use of high-molecular-weight polycaprolactone methacrylate (PCL–M) polymerised high-internal-phase emulsions (polyHIPEs) as a scaffold to support 3D breast cancer (MDA-MB-231) cell culture. We assessed the porosity, interconnectivity, and morphology of the polyHIPEs when varying mixing speed during formation of the emulsion, successfully demonstrating the tunability of these polyHIPEs. An ex ovo chick chorioallantoic membrane assay identified the scaffolds as bioinert, with biocompatible properties within a vascularised tissue. Furthermore, in vitro assessment of cell attachment and proliferation showed promising potential for the use of PCL polyHIPEs to support cell growth. Our results demonstrate that PCL polyHIPEs are a promising material to support cancer cell growth with tuneable porosity and interconnectivity for the fabrication of perfusable 3D cancer models
- …