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25 pags., 14 figs., In their pioneering work demonstrating measurement and full correction of the eye’s optical aberrations, Liang, Williams and Miller, [JOSA A 14, 2884 (1997)] showed improvement in visual performance using adaptive optics (AO). Since then, AO visual simulators have been developed to explore the spatial limits to human vision and as platforms to test non-invasively optical corrections for presbyopia, myopia, or corneal irregularities. These applications have allowed new psychophysics bypassing the optics of the eye, ranging from studying the impact of the interactions of monochromatic and chromatic aberrations on vision to neural adaptation. Other applications address new paradigms of lens designs and corrections of ocular errors. The current paper describes a series of AO visual simulators developed in laboratories around the world, key applications, and current trends and challenges. As the field moves into its second quarter century, new available technologies and a solid reception by the clinical community promise a vigorous and expanding use of AO simulation in years to come., National Eye Institute (P30EY 001319, R01EY014999, R01EY034151); Agencia Estatal de Investigación (PID2019-105684RB-I00, PID2020-115191RB-I00); Vetenskapsrådet (2019-05354); Engineering and Physical Sciences Research Council (G015473/1); Research to Prevent Blindness (N/A Unrestricted funds (UoR)); H2020 Marie SkłodowskaCurie Actions (#675137); Empire State Development’s Division of Science, Technology and Innovation (CEIS #2130C002).

Aim: Human mesenchymal stem cells (hMSC) are multipotent progenitor cells. We propose the optimization of hMSC isolation and recovery using the application of a controlled hypoxic environment. Materials & Methods: We evaluated oxygen, glucose and serum in the recovery of hMSC from bone marrow (BMhMSC). Colony forming units-fibroblastic, cell numbers, tri-lineage differentiation, immunofluorescence and microarray were used to confirm and characterize BMhMSC. Results: In an optimized (2% O2, 4.5 g/l glucose and 5% serum) environment both colony forming units-fibroblastic (p = 0.01) and cell numbers (p = 0.0001) were enhanced over standard conditions. Transcriptional analysis identified differential expression of bone morphogenetic protein 2 (BMP2) and, putatively, chemokine (C-X-C motif) receptor 2 (CXCR2) signaling pathways. Conclusion: We have detailed a potential milestone in the process of refinement of the BMhMSC isolation process.

Fresh frozen bone allograft is available for human recipients after at least 6 months of quarantine at -80 degrees C. It is assumed that cryopreservation without cryoprotectant removes all viable donor cells.We studied the in vitro cell growth from samples of fresh frozen human femoral head allografts after they had been released for patient use, and compared it with cell growth from a control group of fresh cancellous bone specimens from excised femoral heads (8 samples in each group).Cell outgrowths were seen in all of the fresh cancellous bone specimens (100% of replicates, 48 replicates per specimen) but only in a small minority of replicates from 4 of the allograft samples (mean 3.1%). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) investigations revealed that cell outgrowths from both groups contained mRNA for transcription factors Runx2 and Osterix, and also for matrix proteins collagen type I, osteocalcin and bone sialoprotein. This is consistent with the cells being osteoblast-related.This study confirms that fresh frozen human bone allograft cells have the potential to grow in vitro, but the significance of this in recipients is currently unknown.

This study describes the one-step isolation and expansion of marrow stromal cells (MSCs) directly onto the implantable nanofibrous scaffolds. Coverslips were first coated with either aligned or random configurations of poly L,D lactic acid, poly lactic-glycolic acid, and poly-epsilon-caprolactone and then seeded with fresh bone marrow aspirate. Colony-forming units were quantified and the differentiation capacities of the recovered cells were explored. Further optimization was provided by exploring the impact of hyperoxic (21% O(2)) and physiologically approximate (2% O(2)) on cell recovery. Aligned nanofibers in 2% O(2) were identified as being superior for isolation of MSCs. Isolated cells formed colonies following the direction of nanofibers, indicating potential for guided tissue regeneration. The isolated MSCs demonstrated retention of multipotency. These findings offer a rapid, cost-effective method of producing a stem-cell-seeded scaffold for regeneration of multiple tissue types.

Human embryonic stem cells (hESCs) hold great promise therapeutically. In order to deliver on this promise the correct defined conditions for long-term propagation must first be established. Researchers have now provided reports describing the benefits of culturing hESCs in physiologically approximate levels of oxygen. These physiological values fall in the range of 2 to 5% O2. Benefits include reduced spontaneous differentiation, enhanced chromosomal stability and increased clonality. Aims: The aim of our study was to examine the transcriptional consequences of culturing hESCs in physiological normoxia (2% O2) using microarray technology. Methods: Three karyoptically normal hESC lines (H1, H9 and RH1) were examined. At the initiation of this experiment, established hESC lines were redesignated as passage (p) 0 in 21% O2, then bifurcated into 21% O2 and 2% O2, and maintained for a further ten passages at which time samples were again collected. RNA was extracted from all sample points and subjected to microarray analysis using the Affymetrix U133 Plus 2.0 platform. Bioinformatic analysis was performed using dChip and GoStat. Results: We performed grouped analyses of gene expression of early (p0) versus late (p10) air-cultured cells. This revealed relative stability with six (air p0 baseline vs p10 experimental) and one (air p10 baseline vs p0 experimental) gene(s) displaying both greater than twofold and statistically significant upregulation. Conversely, we identified 302 gene upregulations and 56 downregulations when comparing 21% O2 (p0p10) with 2% O2 (p10). These significantly upregulated changes clustered into 82 over-represented and 9 under-represented ontology terms. These terms were indicative of signaling pathways, developmental potential and metabolism. Hierarchical clustering indicated a trend for 2% O2 cultured cells to cluster collectively with reduced heterogeneity when compared with 21% O2 cultured cells. Conclusions: The gene changes associated with 2% O2 culture may be predictive of novel cellular requirements for stable self-renewal, maintenance of pluripotency, and a reduction of hESC-line heterogeneity.

For tissue engineering of load-bearing tissues, such as bone, tendon, cartilage, and cornea, it is critical to generate a highly organized extracellular matrix. The major component of the matrix in these tissues is collagen, which usually forms a highly hierarchical structure with increasing scale from fibril to fiber bundles. These bundles are ordered into a 3D network to withstand forces such as tensile, compressive or shear. To induce the formation of organized matrix and create a mimic body environment for tissue engineering, in particular, tendon tissue engineering, we have fabricated scaffolds with features to support the formation of uniaxially orientated collagen bundles. In addition, mechanical stimuli were applied to stimulate tissue formation and matrix organization. In parallel, we seek a nondestructive tool to monitor the changes within the constructs in response to these external stimulations. Polarizationsensitive optical coherence tomography (PSOCT) is a non-destructive technique that provides functional imaging, and possesses the ability to assess in depth the organization of tissue. In this way, an engineered tissue construct can be monitored on-line, and correlated with the application of different stimuli by PSOCT. We have constructed a PSOCT using a superluminescent diode (FWHM 52nm) in this study and produced two types of tendon constructs. The matrix structural evolution under different mechanical stimulation has been evaluated by the PSOCT. The results in this study demonstrate that PSOCT was a powerful tool enabling us to monitor non-destructively and real time the progressive changes in matrix organization and assess the impact of various stimuli on tissue orientation and growth.

Scaffolds play an important role in the generation of functional tissues using tissue-engineering techniques. To generate highly organised tissue, scaffolds must have specific internal and external architectures. Here, optical coherence tomography (OCT) is exploited to characterise the architectures of various scaffolds, in particular scaffolds which have been fabricated to support the formation of uniaxially orientated collagen bundle for use in tendon tissue engineering. In parallel, a polarisation-sensitive OCT (PSOCT) has been built to assess the collagen fibre organisation in human tendon and monitor the growth of engineering tendon constructs online and non-destructively. The impact of mechanical stimuli on the modulation of tendon tissue formation and organisation was also assessed. It is shown that conventional OCT is capable of characterising scaffold architecture and the pore size, porosity or microchannel dimension can be determined quantitatively and qualitatively. PSOCT generated birefringence images of human tendon and demonstrated that low birefringence images, associated with fewer microstructural variations, correlated to the presence of scar tissue or degenerated tissue; whereas the tissue-engineered tendon exhibited lower degree of birefringence.