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In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5 min) image large sample volumes (1 mm 3 ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm 3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

This study experimentally substantiates that the micromechanical compatibility between cell and substrate is essential for cells to achieve energetically favorable mechanotransduction that directs phenotypic transitions. The argument for this compatibility is based on a thermodynamic model that suggests that the response of cells to their substrate mechanical environment is a consequence of the interchange between forms of energy governing the cell-substrate interaction. Experimental validation for the model has been carried out by investigating the osteogenic differentiation of dental follicle stem cells (DFSCs) seeded on electrospun fibrous scaffolds. Electrospinning of blends containing polycaprolactone (PCL) and silk fibroin (SF) with varying composition of cellulose nanocrystals (CNCs) resulted in three-dimensional (3D) fibrous scaffolds with bimodal distribution of fiber diameter, which provides both macroscopically stiff and microscopically compliant scaffolds for cells without affecting the surface chemical functionality of scaffolds. Atomic force microscopy (AFM) with a colloidal probe and single-cell force spectroscopy were used to characterize cell stiffness and scaffold stiffness on the cellular level, as well as cell-scaffold adhesive interaction (chemical functionality). This study has successfully varied scaffold mechanical properties without affecting their surface chemistry. In vitro tests indicate that the micromechanical compatibility between cells and scaffolds has been significantly correlated with mechanosensitive gene expression markers and osteogenic differentiation markers of DFSCs. The agreement between experimental observations and the thermodynamic model affirms that the cellular response to the mechanical environment, though biological in nature, follows the laws of the energy interchange to achieve its self-regulating behavior. More importantly, this study provides systematic evidence, through extensive and rigorous experimental studies, for the first time that rationalizes that micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

We aim to develop a quantitative viability method that distinguishes individual quiescent from dead cells and is measured in time (ns) as a referenceable, comparable quantity. We demonstrate that fluorescence lifetime imaging of an anionic, fluorescent membrane voltage probe fulfills these requirements for Streptococcus mutans. A random forest machine-learning model assesses whether individual S. mutans can be correctly classified into their original populations: stationary phase (quiescent), heat killed and inactivated via chemical fixation. We compare the results to intensity using three models: lifetime variables (τ 1 , τ 2 and p 1 ), phasor variables (G, S) or all five variables, with the five variable models having the most accurate classification. This initial work affirms the potential for using fluorescence lifetime of a membrane voltage probe as a viability marker for quiescent bacteria, and future efforts on other bacterial species and fluorophores will help refine this approach., (© 2021 Wiley-VCH GmbH.)

Nanofiber scaffolds can induce osteogenic differentiation and cell morphology alterations of human bone marrow stromal cells (hBMSCs) without introduction of chemical cues. In this study, we investigate the predictive power of day 1 cell morphology, quantified by a machine learning based method, as an indicator of osteogenic differentiation modulated by nanofiber density. Nanofiber scaffolds are fabricated via electrospinning. Microscopy, quantitative image processing and clustering analysis are used to systematically quantify scaffold properties as a function of fiber density. hBMSC osteogenic differentiation potential is evaluated after 14 days using osteogenic marker gene expression and after 50 days using calcium mineralization, showing enhanced osteogenic differentiation with an increase in nanofiber density. Cell morphology measurements at day 1 successfully predict differentiation potential when analyzed with the support vector machine (SVM)/supercell tools previously developed and trained on cells from a different donor. A correlation is observed between differentiation potential and cell morphology, demonstrating sensitivity of the morphology measurement to varying degrees of differentiation potential. To further understand how nanofiber density determines hBMSC morphology, both full 3-D morphology measurements as well as other measurements of the 2-D projected morphology are investigated in this study. To achieve predictive power on hBMSC osteogenic differentiation, at least two morphology metrics need to be considered together for each cell, with the majority of metric pairs including one 3-D morphology metric. Analysis of the local nanofiber structure surrounding each cell reveals a correlation with single-cell morphology and indicates that the osteogenic differentiation phenotype may be predictive at the single-cell level., (Published by Elsevier Ltd.)

Despite their great promise as fluorescent biological probes and sensors, the structure and dynamics of Ag complexes derived from single stranded DNA (ssDNA) are less understood than their double stranded counterparts. In this work, we seek new insights into the structure of single AgNssDNA clusters using analytical ultracentrifugation (AUC), nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular dynamics simulations (MD) of a fluorescent (AgNssDNA)8+ nanocluster. The results suggest that the purified (AgNssDNA)8+ nanocluster is a mixture of predominantly Ag15 and Ag16 species that prefer two distinct long-lived conformational states: one extended, the other approaching spherical. However, the ssDNA strands within these clusters are highly mobile. Ag(i) interacts preferentially with the nucleobase rather than the phosphate backbone, causing a restructuring of the DNA strand relative to the bare DNA. Infrared spectroscopy and MD simulations of (AgNssDNA)8+ and model nucleic acid homopolymers suggest that Ag(i) has a higher affinity for cytosine over guanine bases, little interaction with adenine, and virtually none with thymine. Ag(i) shows a tendency to interact with cytosine N3 and O2 and guanine N7 and O6, opening the possibility for a Ag(i)-base bifurcated bond to act as a nanocluster nucleation and strand stabilizing site. This work provides valuable insight into nanocluster structure and dynamics which drive stability and optical properties, and additional studies using these types of characterization techniques are important for the rational design of single stranded AgDNA nanocluster sensors.

Cell morphology has been identified as a potential indicator of stem cell response to biomaterials. However, determination of cell shape phenotype in biomaterials is complicated by heterogeneous cell populations, microenvironment heterogeneity, and multi-parametric definitions of cell morphology. To associate cell morphology with cell-material interactions, we developed a shape phenotyping framework based on support vector machines. A feature selection procedure was implemented to select the most significant combination of cell shape metrics to build classifiers with both accuracy and stability to identify and predict microenvironment-driven morphological differences in heterogeneous cell populations. The analysis was conducted at a multi-cell level, where a "supercell" method used average shape measurements of small groups of single cells to account for heterogeneous populations and microenvironment. A subsampling validation algorithm revealed the range of supercell sizes and sample sizes needed for classifier stability and generalization capability. As an example, the responses of human bone marrow stromal cells (hBMSCs) to fibrous vs flat microenvironments were compared on day 1. Our analysis showed that 57 cells (grouped into supercells of size 4) are the minimum needed for phenotyping. The analysis identified that a combination of minor axis length, solidity, and mean negative curvature were the strongest early shape-based indicator of hBMSCs response to fibrous microenvironment., (Copyright © 2016. Published by Elsevier Ltd.)

The focus on creating tissue engineered constructs of clinically relevant sizes requires new approaches for monitoring construct health during tissue development. A few key requirements are that the technology be in situ, non-invasive, and provide temporal and spatial information. In this work, we demonstrate that optical coherence microscopy (OCM) can be used to assess cell viability without the addition of exogenous probes in three-dimensional (3D) tissue scaffolds maintained under standard culture conditions. This is done by collecting time-lapse images of speckle generated by sub-cellular features. Image cross-correlation is used to calculate the number of features the final image has in common with the initial image. If the cells are live, the number of common features is low. The number of common features approaches 100% if the cells are dead. In control experiments, cell viability is verified by the addition of a two-photon fluorescence channel to the OCM. Green fluorescent protein transfected human bone marrow stromal cells cultured in a transparent poly(ethylene glycol) tetramethacrylate hydrogel scaffold is used as the control system. Then, the utility of this approach is demonstrated by determining L929 fibroblast cell viability in a more challenging matrix, collagen, an optical scatterer. These results demonstrate a new technique for in situ mapping of single cell viability without any exogenous probes that is capable of providing continuous monitoring of construct health., (Published by Elsevier Ltd.)

Objective: This investigation was to generate (1) guidelines for designing a tensometer that satisfies the necessary accuracy and sensitivity requirements for measuring polymerization stress (PS), and (2) a formula for calculating PS. Polymerization stress remains one of the most critical properties of polymeric dental materials, yet methods that can accurately quantify PS have been limited in part due to the complexity of polymerization, and in part due to the instrumentation itself., Method: In this study, we performed analytical and finite element analyses on a cantilever-beam based tensometer that is used to evaluate shrinkage stresses during the polymerization of dental restorative composites., Results: The PS generated by a commercial dental composite determined using our new tensometer agrees with the predicted trend when the beam length and/or specimen height is varied., Significance: This work demonstrates the importance of beam dimension and component relative rigidity to the accuracy of PS evaluation. An analytical solution is also derived for the vertical beam deflection, which can be used for any combination of bending and shearing to properly calculate the PS. In addition, an easy-to-conduct calibration procedure is provided that is desirable for periodic tensometer recalibration., (Published by Elsevier Ltd.)

When culturing cells on flexible surfaces, it is important to consider extracellular matrix treatments that will remain on the surface under mechanical strain. Here we investigate differences in laminin deposited on oxidized polydimethylsiloxane (PDMS) with plasma treatment (plasma-only) vs. plasma and aminopropyltrimethoxysilane treatment (silane-linked). We use specular X-ray reflectivity (SXR), transmission electron microscopy (TEM), and immunofluorescence to probe the quantity and uniformity of laminin. The surface coverage of laminin is approximately 45% for the plasma-only and 50% for the silane-linked treatment as determined by SXR. TEM and immunofluorescence reveal additional islands of laminin aggregates on the plasma-only PDMS compared with the relatively smooth and uniform silane-linked laminin surface. We also examine laminin retention under strain and vascular smooth muscle cell viability and proliferation under static and strain conditions. Equibiaxial stretching of the PDMS surfaces shows greatly improved retention of the silane-linked laminin over plasma-only. There are significantly more cells on the silane-linked surface after 4 days of equibiaxial strain., (Published by Elsevier Ltd.)

Many methods exist in the literature to modify surfaces with extracellular matrix (ECM) proteins prior to cell seeding. However, there are few studies that systematically characterize and compare surface properties and cell response results among modification methods that use different bonding mechanisms. In this work, we compare cell response and physical characterization results from fibronectin or laminin attached to polydimethylsiloxane (PDMS) elastomer surfaces by physical adsorption, chemisorption, and covalent attachment to determine the best method to modify a deformable surface. We evaluate modification methods based on completeness and uniformity of coverage, surface roughness, and hydrophilicity of attached ECM protein. Smooth muscle cell adhesion, proliferation, morphology, and phenotype were also evaluated. We found that chemisorption methods resulted in higher amounts of protein attachment than physical adsorption and covalent bonding of the ECM proteins. Cell response to protein-modified surfaces was similar with respect to cell adhesion, area, aspect ratio, and phenotype. When all the data are considered, the chemisorption methods, most notably silane_70, provide the best surface properties and highest cell proliferation., ((c) 2009 Wiley Periodicals, Inc.)

This paper describes a set of tools for performing measurements of objects in a virtual reality based immersive visualization environment. These tools enable the use of the immersive environment as an instrument for extracting quantitative information from data representations that hitherto had be used solely for qualitative examination. We provide, within the virtual environment, ways for the user to analyze and interact with the quantitative data generated. We describe results generated by these methods to obtain dimensional descriptors of tissue engineered medical products. We regard this toolbox as our first step in the implementation of a virtual measurement laboratory within an immersive visualization environment.

The capability to image real time cell/material interactions in a three-dimensional (3D) culture environment will aid in the advancement of tissue engineering. This paper describes a perfusion flow bioreactor designed to hold tissue engineering scaffolds and allow for in situ imaging using an upright microscope. The bioreactor can hold a scaffold of desirable thickness for implantation (>2 mm). Coupling 3D culture and perfusion flow leads to the creation of a more biomimetic environment. We examined the ability of the bioreactor to maintain cell viability outside of an incubator environment (temperature and pH stability), investigated the flow features of the system (flow induced shear stress), and determined the image quality in order to perform time-lapsed imaging of two-dimensional (2D) and 3D cell culture. In situ imaging was performed on 2D and 3D, culture samples and cell viability was measured under perfusion flow (2.5 mL/min, 0.016 Pa). The visualization of cell response to their environment, in real time, will help to further elucidate the influences of biomaterial surface features, scaffold architectures, and the influence of flow induced shear on cell response and growth of new tissue., ((c) 2006 Wiley Periodicals, Inc.)

A novel technique is described to investigate buried polymer/sizing/substrate interfacial regions, in situ, by localizing a fluorescent probe molecule in the sizing layer. Epoxy functional silane coupling agent multilayers were deposited on glass microscope cover slips and doped with small levels of a fluorescently labeled silane coupling agent (FLSCA). The emission of the grafted FLSCA was dependent on the silane layer thickness, showing blue-shifted emission with decreasing thickness. The fluorescent results suggest that thinner layers were more tightly bound to the glass surface. The layers were also characterized by scanning electron microscopy, contact angle, and thermogravimetric analysis (TGA). When the FLSCA-doped silane layers were immersed in epoxy resin, a blue shift in emission occurred during resin cure, indicating the potential to study interfacial chemistry, in situ. Thicker silane layers exhibited smaller fluorescence shifts during cure, suggesting incomplete resin penetration into the thickest silane layers.

Objectives: The aim of this study was to investigate the feasibility of using the microbond test (MBT) to probe the durability of the bond between a polymerized dental resin with differently silanized E-glass fibers., Methods: The E-glass fibers were silanized with equivalent amounts of two types of acrylic-silane coupling agents: 3-methacryloxypropyltrimethoxysilane (MPTMS) and 10-methacryloxydecyltrimethoxysilane (MDTMS), a more hydrophobic silane coupling agent than MPTMS. Unsilanized E-glass fibers were used as the control. Microdroplets of a photo-activated dental resin were applied on the fiber and photocured with visible light irradiation (470 nm). Subsequently, the specimens were tested in shear after 24h storage in air at 23 degrees C or water at 60 degrees C., Results: The mean interfacial shear strength (tau) and the standard deviation in MPa for the three systems in 23 degrees C in air (n>7) were: 33.8(10.1), 33.7(8.9) and 15.3(4.2) for the MPTMS silanized, MDTMS silanized, and unsilanized fibers, respectively. When the three types of fibers were first exposed to 60 degrees C water for 24h prior to having the microdroplets of the resin bonded to them, the strength values of the MDTMS silanized fibers and the control fibers remained essentially unchanged at (n> or =7) 31.8(7.7) and 17.5(4.9)MPa respectively; the MPTMS specimens showed a significant decrease to 15.8(4.8)MPa. Similar trends were observed when the fibers had microdroplets of the resin bonded to them prior to aqueous exposure., Significance: These results indicate that the microbond test has the sensitivity to measure changes at the interface between polymerized dental resins and variously silanized E-glass fibers. It appears that surface modification of the fibers with the more hydrophobic silane coupling agent MDTMS promotes enhanced resistance to degradation from exposure to water. The microbond test has the potential for studying dental adhesion involving small bonded areas under a variety of conditions with different adhesive systems and substrates.

Silane coupling agents are commonly applied to glass fibers to promote fiber/resin adhesion and enhance durability in composite parts. In this study, a coupling agent multilayer on glass was doped with trace levels of the dimethylaminonitrostilbene (DMANS) fluorophore. The fluorophore was immobilized on the glass surface by tethering the molecule to a triethoxy silane coupling agent, creating the DMANS/silane coupling agent molecule (DMSCA). DMSCA was then diluted with commonly used coupling agents and grafted to a glass microscope coverslip to create a model composite interface. A 53-nm blue shift in fluorescence from the immobilized DMSCA can be followed during cure of an epoxy resin overlayer, giving this technique potential to monitor the properties of the fiber/resin interface during composite processing. Contact angle measurements on these coupling agent layers were similar in the presence or absence of the DMSCA molecule, suggesting that trace levels of the fluorescent probe did not affect the structure of the layer. The immobilized DMSCA molecule behaved similarly to the DMANS precursor in solution. Both showed longer wavelength fluorescence in more polar environments. Copyright 2000 Academic Press.