We present a novel indentation method for characterizing the viscoelastic properties of alginate and agarose hydrogel based constructs, which are often used as a model system of soft biological tissues. A sensitive long working distance microscope was used for measuring the time-dependent deformation of the thin circular hydrogel membranes under a constant load. The deformation of the constructs was measured laterally. The elastic modulus as a function of time can be determined by a large deformation theory based on Mooney–Rivlin elasticity. A viscoelastic theory, Zener model, was applied to correlate the time-dependent deformation of the constructs with various gel concentrations, and the creep parameters can therefore be quantitatively estimated. The value of Young's modulus was shown to increase in proportion with gel concentration. This finding is consistent with other publications. Our results also showed the great capability of using the technique to measure gels with incorporated corneal stromal cells. This study demonstrates a novel and convenient technique to measure mechanical properties of hydrogel in a non-destructive, online and real-time fashion. Thus this novel technique can become a valuable tool for soft tissue engineering.

Tendon tissue engineering requires the generation of a uniaxially orientated collagen type I matrix with several organization scales that confer mechanical functionality upon the tendon. A combination of factors in a dose- and time-dependent manner, such as growth factors and mechanical environment, may be the key to an in vitro–engineered tendon. To define the progress of tissue development within a scaffold, on-line systems need to be applied to monitor the newly generated matrix. To address this challenge, we designed a new porous chitosan scaffold with microchannels (diameter: 250 μm), which allows primary porcine tenocytes to proliferate in a bundle-like structure. The cell proliferation and extracellular matrix (ECM) production within the microchannels were successfully assessed under sterile conditions using optical coherence tomography (OCT). A semi-quantitative method that calculated the microchannel occupation ratio (the degree of cell proliferation and tissue turnover based on the total backscattered intensity in the microchannels) was developed. We further investigated the effect of different culture conditions on tendon cell matrix formation. Using a perfusion bioreactor, we demonstrated how fluid flow can increase (p < 1e⁻³) ECM production within the microchannels significantly more than static culture. Our study illustrates how using a guiding scaffold in combination with the fast and non-destructive assessment of the microstructure using OCT allows discrimination between the parameters affecting the production and the organization of the ECM.

Corneal structure is highly organized and unified in architecture with structural and functional integration which mediates transparency and vision. Disease and injury are the second most common cause of blindness affecting over 10 million people worldwide. Ninety percent of blindness is permanent due to scarring and vascularization. Scarring caused via fibrotic cellular responses, heals the tissue, but fails to restore transparency. Controlling keratocyte activation and differentiation are key for the inhibition and prevention of fibrosis. Ophthalmic surgery techniques are continually developing to preserve and restore vision but corneal regression and scarring are often detrimental side effects and long term continuous follow up studies are lacking or discouraging. Appropriate corneal models may lead to a reduced need for corneal transplantation as presently there are insufficient numbers or suitable tissue to meet demand. Synthetic optical materials are under development for keratoprothesis although clinical use is limited due to implantation complications and high rejection rates. Tissue engineered corneas offer an alternative which more closely mimic the morphological, physiological and biomechanical properties of native corneas. However, replication of the native collagen fiber organization and retaining the phenotype of stromal cells which prevent scar-like tissue formation remains a challenge. Careful manipulation of culture environments are under investigation to determine a suitable environment that simulates native ECM organization and stimulates keratocyte migration and generation.

Monitoring cell profiles in 3D porous scaffolds presents a major challenge in tissue engineering. In this study, we investigate optical coherence tomography (OCT) as an imaging modality to monitor non-invasively both structures and cells in engineered tissue constructs. We employ time-domain OCT to visualize macro-structural morphology, and whole-field optical coherence microscopy to delineate the morphology of cells and constructs in a developing in vitro engineered bone tissue. The results show great potential for the use of OCT in non-invasive monitoring of cellular activities in 3D developing engineered tissues.

Control and maintenance of the keratocyte phenotype is vital to developing in vitro tissue engineered strategies for corneal repair. In this study the influence of topographical and chemical cues on the mechanical, phenotypical and genotypical behaviour of adult human derived corneal stromal (AHDCS) cells in three dimensional (3D) multi‐layered organised constructs is examined. Topographical cues are provided via multiple aligned electrospun nanofiber meshes, which are arranged orthogonally throughout the constructs and are capable of aligning individual cells and permitting cell migration between the layers. The influence of chemical cues is examined using different supplements in culture media. A non‐destructive indentation technique and optical coherence tomography are used to determine the matrix elasiticity (elastic modulus) and dimensional changes, respectively. These measurements were indicative of changes in cell phenotype from contractile fibroblasts to quiescent keratocytes over the duration of the experiment and corroborated by qPCR. Constructs containing nanofibers have a higher initial modulus, reduced contraction and organised cell orientation compared to those without nanofibers. Cell‐seeded constructs cultured in serum‐containing media increased in modulus throughout the culture period and underwent significantly more contraction than constructs cultured in serum‐free and insulin‐containing media. This implies that the growth factors present in serum promote a fibroblast‐like phenotype; qPCR data further validates these observations. These results indicate that the synergistic effect of nanofibers and serum‐free media plus insulin supplementation provide the most suitable topographical and chemical environment for reverting corneal fibroblasts to a keratocyte phenotype in a 3D construct.

It is clear that nanofibrous structures can be used as tools for many applications. It is already known that electrospinning is a highly versatile method of producing nanofibres and recent developments in the technique of electrospinning have led to the development of aligned nanofibres and biphasic, core-sheath fibres which can be used to encapsulate different materials from molecules to cells. Natural extracellular matrix (ECM) contains fibres in both micro and nano-scales and provides a structural scaffold which allows cells to localize, migrate, proliferate and differentiate. Polymer nanofibres can provide the structural cues of ECM. However, current literature gives new hope to further functionalising polymeric nanofibres by using them for drug delivery devices and improving their design to improve control of delivery. By encapsulating active agents within nanofibres (multifunctional nanofibres), a degree of control can be exerted over the release of encapsulated agents and therefore, the behaviour of cells can be manipulated for developing effective therapies and is extremely encouraging in the tissue engineering field by combining factors like fibre diameter, alignment and chemicals in new ways. Such multifunctional nanofibre-based systems are already being investigated in vivo. Experiments have shown the significant potential for treatments of disease and engineering of neural and bone tissues. Further, phase III clinical trials of nanofibrous patches for applications in wound treatment were encouraging. Hopefully, clinical applications of these drug delivery devices will follow, to enhance regenerative medicine applications.