Development, structure, and bioengineering of the human corneal stroma: A review of collagen-based implants.

Bio-engineering technologies are currently used to produce biomimetic artificial corneas that should present structural, chemical, optical, and biomechanical properties close to the native tissue. These properties are mainly supported by the corneal stroma which accounts for 90% of corneal thickness and is mainly made of collagen type I. The stromal collagen fibrils are arranged in lamellae that have a plywood-like organization. The fibril diameter is between 25 and 35 nm and the interfibrillar space about 57 nm. The number of lamellae in the central stroma is estimated to be 300. In the anterior part, their size is 10-40 µm. They appear to be larger in the posterior part of the stroma with a size of 60-120 µm. Their thicknesses also vary from 0.2 to 2.5 µm. During development, the acellular corneal stroma, which features a complex pattern of organization, serves as a scaffold for mesenchymal cells that invade and further produce the cellular stroma. Several pathways including Bmp4, Wnt/ß-catenin, Notch, retinoic acid, and TGF-ß, in addition to EFTFs including the mastering gene Pax-6, are involved in corneal development. Besides, retinoic acid and TGF- ß seem to have a crucial role in the neural crest cell migration in the stroma. Several technologies can be used to produce artificial stroma. Taking advantage of the liquid-crystal properties of acid-soluble collagen, it is possible to produce transparent stroma-like matrices with native-like collagen I fibrils and plywood-like organization, where epithelial cells can adhere and proliferate. Other approaches include the use of recombinant collagen, cross-linkers, vitrification, plastically compressed collagen or magnetically aligned collagen, providing interesting optical and mechanical properties. These technologies can be classified according to collagen type and origin, presence of telopeptides and native-like fibrils, structure, and transparency. Collagen matrices feature transparency >80% for the appropriate 500-µm thickness. Non-collagenous matrices made of biopolymers including gelatin, silk, or fish scale have been developed which feature interesting properties but are less biomimetic. These bioengineered matrices still need to be colonized by stromal cells to fully reproduce the native stroma.

Implementation of artifact-free circular dichroism SHG imaging of collagen.

Second harmonic generation (SHG) enables in situ imaging of fibrillar collagen architecture in connective tissues. Recently, Circular Dichroism SHG (CD-SHG) microscopy has been implemented to take advantage of collagen chirality to improve 3D visualization. It measures the normalized difference in the SHG signal obtained upon excitation by left versus right circular polarizations. However, CD-SHG signal is not well characterized yet, and quite different CD-SHG values are reported in the literature. Here, we identify two major artifacts that may occur in CD-SHG experiments and we demonstrate that thorough optimization and calibration of the experimental setup are required for CD-SHG imaging. Notably it requires a careful calibration of the incident circular polarizations and a perfect mechanical stabilization of the microscope stage. Finally, we successfully record CD-SHG images in human cornea sections and confirm that this technique efficiently reveals collagen fibrils oriented out of the focal plane.

Stabilization of Collagen Fibrils by Gelatin Addition: A Study of Collagen/Gelatin Dense Phases.

Collagen and its denatured form, gelatin, are biopolymers of fundamental interest in numerous fields ranging from living tissues to biomaterials, food, and cosmetics. This study aims at characterizing mixtures of those biopolymers at high concentrations (up to 100 mg·mL-1) at which collagen has mesogenic properties. We use a structural approach combining polarization-resolved multiphoton microscopy, polarized light microscopy, magnetic resonance imaging, and transmission electron microscopy to analyze gelatin and collagen/gelatin dense phases in their sol and gel states from the macroscopic to the microscopic scale. We first report the formation of a lyotropic crystal phase of gelatin A and show that gelatin must structure itself in particles to become mesogenic. We demonstrate that mixtures of collagen and gelatin phase segregate, preserving the setting of the pure collagen mesophase at a gelatin ratio of up to 20% and generating a biphasic fractal sample at all tested ratios. Moreover, differential scanning calorimetric analysis shows that each protein separates into two populations. Both populations of gelatins are stabilized by the presence of collagen, whereas only one population of collagen molecules is stabilized by the presence of gelatin, most probably those at the interface of the fibrillated microdomains and of the gelatin phase. Although further studies are needed to fully understand the involved mechanism, these new data should have a direct impact on the bioengineering of those two biopolymers.

Cell viability and shock wave amplitudes in the endothelium of porcine cornea exposed to ultrashort laser pulses.

PURPOSE: Some forms of keratoplasty assisted by ultrashort-pulse lasers require performing laser cuts close to the endothelium, which requires the knowledge of "safe" values concerning incision depth and pulse energy preserving endothelial cell viability. Our study aims to determine the thresholds for cell death in porcine corneas exposed to ultrashort laser pulses, in terms of laser pulse energy and nearness of the impacts to the endothelium. METHODS: Using a laboratory laser set-up, lamellar cuts were induced while varying pulse energies and distances from the endothelium. A fluorescent staining protocol was used to determine the percentage of surviving endothelial cells. Numerical simulations of the Euler equations for compressible fluids provided pressure level and axial and radial pressure gradient estimates at the endothelium. RESULTS: Ninety percent of the endothelial cells survived when using 16.5 µJ pulses no closer than 200 µm to the endothelium, or pulses not exceeding 2 µJ at a distance of 50 µm. The comparison of the observed percentage of surviving cells with the estimates of the shock wave amplitudes and gradients generated by the laser pulses yielded cell death thresholds at amplitudes in the megapascal range, or gradients of the order of 108 Pa/m. CONCLUSIONS: Our results provide limits in terms of pulse energy and distance of the incision from the endothelium within which endothelial cell viability is preserved. Current forms of corneal laser surgery are compatible with these limits. However, these limits will need to be considered for the development of future laser routines working in close proximity to the endothelium.

Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy.

This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules.

Theoretical, numerical and experimental study of geometrical parameters that affect anisotropy measurements in polarization-resolved SHG microscopy.

Polarization-resolved second harmonic generation (P-SHG) microscopy is an efficient imaging modality for in situ observation of biopolymers structure in tissues, providing information about their mean in-plane orientation and their molecular structure and 3D distribution. Nevertheless, P-SHG signal build-up in a strongly focused regime is not throroughly understood yet, preventing reliable and reproducible measurements. In this study, theoretical analysis, vectorial numerical simulations and experiments are performed to understand how geometrical parameters, such as excitation and collection numerical apertures and detection direction, affect P-SHG imaging in homogeneous collagen tissues. A good agreement is obtained in tendon and cornea, showing that detection geometry significantly affects the SHG anisotropy measurements, but not the measurements of collagen in-plane orientation.

Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication.

Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks.

Determination of collagen fiber orientation in histological slides using Mueller microscopy and validation by second harmonic generation imaging.

We studied the azimuthal orientations of collagen fibers in histological slides of uterine cervical tissue by two different microscopy techniques, namely Mueller polarimetry (MP) and Second Harmonic Generation (SHG). SHG provides direct visualization of the fibers with high specificity, which orientations is then obtained by suitable image processing. MP provides images of retardation (among other polarimetric parameters) due to the optical anisotropy of the fibers, which is enhanced by Picrosirius Red staining. The fiber orientations are then assumed to be those of the retardation slow axes. The two methods, though fully different from each other, provide quite similar maps of average fiber orientations. Overall, our results confirm that MP microscopy provides reliable images of dominant fiber orientations at a much lower cost that SHG, which remains the "gold standard" for specific imaging of collagen fibers using optical microscopy.

Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics.

The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects.

The Balance Between Shear Flow and Extracellular Matrix in Ovarian Cancer-on-Chip.

Ovarian cancer is the most lethal gynecologic cancer in developed countries. In the tumor microenvironment, the extracellular matrix (ECM) and flow shear stress are key players in directing ovarian cancer cells invasion. Artificial ECM models based only on ECM proteins are used to build an ovarian tumor-on-chip to decipher the crosstalk between ECM and shear stress on the migratory behavior and cellular heterogeneity of ovarian tumor cells. This work shows that in the shear stress regime of the peritoneal cavity, the ECM plays a major role in driving individual or collective ovarian tumor cells migration. In the presence of basement membrane proteins, migration is more collective than on type I collagen regardless of shear stress. With increasing shear stress, individual cell migration is enhanced; while, no significant impact on collective migration is measured. This highlights the central position that ECM and flow shear stress should hold in in vitro ovarian cancer models to deepen understanding of cellular responses and improve development of ovarian cancer therapeutic platforms. In this frame, adding flow provides significant improvement in biological relevance over the authors' previous work. Further steps for enhanced clinical relevance require not only multiple cell lines but also patient-derived cells and sera.

Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy.

A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1-3 µm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500-600 µm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior-superior axis in the anterior stroma and along the nasal-temporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues.

Impact of corpus callosum fiber tract crossing on polarimetric images of human brain histological sections: ex vivo studies in transmission configuration.

Significance: Imaging Mueller polarimetry is capable to trace in-plane orientation of brain fiber tracts by detecting the optical anisotropy of white matter of healthy brain. Brain tumor cells grow chaotically and destroy this anisotropy. Hence, the drop in scalar retardance values and randomization of the azimuth of the optical axis could serve as the optical marker for brain tumor zone delineation. Aim: The presence of underlying crossing fibers can also affect the values of scalar retardance and the azimuth of the optical axis. We studied and analyzed the impact of fiber crossing on the polarimetric images of thin histological sections of brain corpus callosum. Approach: We used the transmission Mueller microscope for imaging of two-layered stacks of thin sections of corpus callosum tissue to mimic the overlapping brain fiber tracts with different fiber orientations. The decomposition of the measured Mueller matrices was performed with differential and Lu-Chipman algorithms and completed by the statistical analysis of the maps of scalar retardance, azimuth of the optical axis, and depolarization. Results: Our results indicate the sensitivity of Mueller polarimetry to different spatial arrangement of brain fiber tracts as seen in the maps of scalar retardance and azimuth of optical axis of two-layered stacks of corpus callosum sections The depolarization varies slightly (<15%) with the orientation of the optical axes in both corpus callosum stripes, but its value increases by 2.5 to 3 times with the stack thickness. Conclusions: The crossing brain fiber tracts measured in transmission induce the drop in values of scalar retardance and randomization of the azimuth of the optical axis at optical path length of 15 µm. It suggests that the presence of nerve fibers crossing within the depth of few microns will be also detected in polarimetric maps of brain white matter measured in reflection configuration.

Elastic fiber alterations and calcifications in calcific uremic arteriolopathy.

Calcific uremic arteriolopathy (CUA) is a severely morbid disease, affecting mostly dialyzed end-stage renal disease (ESRD) patients, associated with calcium deposits in the skin. Calcifications have been identified in ESRD patients without CUA, indicating that their presence is not specific to the disease. The objective of this retrospective multicenter study was to compare elastic fiber structure and skin calcifications in ESRD patients with CUA to those without CUA using innovative structural techniques. Fourteen ESRD patients with CUA were compared to 12 ESRD patients without CUA. Analyses of elastic fiber structure and skin calcifications using multiphoton microscopy followed by machine-learning analysis and field-emission scanning electron microscopy coupled with energy dispersive X-ray were performed. Elastic fibers specifically appeared fragmented in CUA. Quantitative analyses of multiphoton images showed that they were significantly straighter in ESRD patients with CUA than without CUA. Interstitial and vascular calcifications were observed in both groups of ESRD patients, but vascular calcifications specifically appeared massive and circumferential in CUA. Unlike interstitial calcifications, massive circumferential vascular calcifications and elastic fibers straightening appeared specific to CUA. The origins of such specific elastic fiber's alteration are still to be explored and may involve relationships with ischemic vascular or inflammatory processes.

Polarization-resolved second-harmonic generation in tendon upon mechanical stretching.

Collagen is a triple-helical protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical and physical properties of most organs. Second-harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. In this article, we use a multiscale nonlinear optical formalism to bring theoretical evidence that anisotropy of polarization-resolved SHG mostly reflects the micrometer-scale disorder in the collagen fibril distribution. Our theoretical expectations are confirmed by experimental results in rat-tail tendon. To that end, we report what to our knowledge is the first experimental implementation of polarization-resolved SHG microscopy combined with mechanical assays, to simultaneously monitor the biomechanical response of rat-tail tendon at macroscopic scale and the rearrangement of collagen fibrils in this tissue at microscopic scale. These experiments bring direct evidence that tendon stretching corresponds to straightening and aligning of collagen fibrils within the fascicle. We observe a decrease in the SHG anisotropy parameter when the tendon is stretched in a physiological range, in agreement with our numerical simulations. Moreover, these experiments provide a unique measurement of the nonlinear optical response of aligned fibrils. Our data show an excellent agreement with recently published theoretical calculations of the collagen triple helix hyperpolarizability.

In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy.

We demonstrate multimodal nonlinear optical imaging of historical artifacts by combining Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (2PEF) microscopies. We first identify the nonlinear optical response of materials commonly encountered in coatings of cultural heritage artifacts by analyzing one- and multi-layered model samples. We observe 2PEF signals from cochineal lake and sandarac and show that pigments and varnish films can be discriminated by exploiting their different emission spectral ranges as in luminescence linear spectroscopy. We then demonstrate SHG imaging of a filler, plaster, composed of bassanite particles which exhibit a non centrosymmetric crystal structure. We also show that SHG/2PEF imaging enables the visualization of wood microstructure through typically 60 µm-thick coatings by revealing crystalline cellulose (SHG signal) and lignin (2PEF signal) in the wood cell walls. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin. SHG/2PEF imaging thus appears as a promising non-destructive and contactless tool for in situ 3D investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale.

Three-Dimensional Collagen Topology Shapes Cell Morphology, beyond Stiffness.

Cellular heterogeneity is associated with many physiological processes, including pathological ones, such as morphogenesis and tumorigenesis. The extracellular matrix (ECM) is a key player in the generation of cellular heterogeneity. Advances in our understanding rely on our ability to provide relevant in vitro models. This requires obtainment of the characteristics of the tissues that are essential for controlling cell fate. To do this, we must consider the diversity of tissues, the diversity of physiological contexts, and the constant remodeling of the ECM along these processes. To this aim, we have fabricated a library of ECM models for reproducing the scaffold of connective tissues and the basement membrane by using different biofabrication routes based on the electrospinning and drop casting of biopolymers from the ECM. Using a combination of electron microscopy, multiphoton imaging, and AFM nanoindentation, we show that we can vary independently protein composition, topology, and stiffness of ECM models. This in turns allows one to generate the in vivo complexity of the phenotypic landscape of ovarian cancer cells. We show that, while this phenotypic shift cannot be directly correlated with a unique ECM feature, the three-dimensional collagen fibril topology patterns cell shape, beyond protein composition and stiffness of the ECM. On this line, this work is a further step toward the development of ECM models recapitulating the constantly remodeled environment that cells face and thus provides new insights for cancer model engineering and drug testing.

Quantitative structural imaging of keratoconic corneas using polarization-resolved SHG microscopy.

The human cornea is mainly composed of collagen fibrils aligned together within stacked lamellae. This lamellar structure can be affected in pathologies such as keratoconus, which is characterized by progressive corneal thinning and local steepening. In this study, we use polarization-resolved second harmonic generation (P-SHG) microscopy to characterize 8 control and 6 keratoconic human corneas. Automated processing of P-SHG images of transverse sections provides the collagen orientation in every pixel with sub-micrometer resolution. Series of P-SHG images recorded in the most anterior region of the stroma evidence sutural lamellae inclined at 22° ± 5° to the corneal surface, but show no significant difference between control and keratoconic corneas. In contrast, series of P-SHG images acquired along the full thickness of the stroma show a loss of order in the lamellar structure of keratoconic corneas, in agreement with their defective mechanical properties. This structural difference is analyzed quantitatively by computing the entropy and the orientation index of the collagen orientation distribution and significant differences are obtained along the full thickness of the stroma. This study shows that P-SHG is an effective tool for automatic quantitative analysis of structural defects of human corneas and should be applied to other collagen-rich tissues.

Noninvasive quantitative assessment of collagen degradation in parchments by polarization-resolved SHG microscopy.

Nondestructive and noninvasive investigation techniques are highly sought-after to establish the degradation state of historical parchments, which is up to now assessed by thermal techniques that are invasive and destructive. We show that advanced nonlinear optical (NLO) microscopy enables quantitative in situ mapping of parchment degradation at the micrometer scale. We introduce two parameters that are sensitive to different degradation stages: the ratio of two-photon excited fluorescence to second harmonic generation (SHG) signals probes severe degradation, while the anisotropy parameter extracted from polarization-resolved SHG measurements is sensitive to early degradation. This approach is first validated by comparing NLO quantitative parameters to thermal measurements on artificially altered contemporary parchments. We then analyze invaluable parchments from the Middle Ages and show that we can map their conservation state and assess the impact of a restoration process. NLO quantitative microscopy should therefore help to identify parchments most at risk and optimize restoration methods.

Rapid Evaluation of Novel Therapeutic Strategies Using a 3D Collagen-Based Tissue-Like Model.

2D cell cultures are commonly used to rapidly evaluate the therapeutic potential of various treatments on living cells. However, the effects of the extracellular matrix (ECM) including the 3D arrangement of cells and the complex physiology of native environment are missing, which makes these models far from in vivo conditions. 3D cell models have emerged in preclinical studies to simulate the impact of the ECM and partially bridge the gap between monolayer cultures and in vivo tissues. To date, the difficulty to handle the existing 3D models, the cost of their production and their poor reproducibility have hindered their use. Here, we present a reproducible and commercially available "3D cell collagen-based model" (3D-CCM) that allows to study the influence of the matrix on nanoagent uptake and radiation effects. The cell density in these samples is homogeneous. The oxygen concentration in the 3D-CCM is tunable, which opens the opportunity to investigate hypoxic effects. In addition, thanks to the intrinsic properties of the collagen, the second harmonic imaging microscopy may be used to probe the whole volume and visualize living cells in real-time. Thus, the architecture and composition of 3D-CCMs as well as the impact of various therapeutic strategies on cells embedded in the ECM is observed directly. Moreover, the disaggregation of the collagen matrix allows recovering of cells without damaging them. It is a major advantage that makes possible single cell analysis and quantification of treatment effects using clonogenic assay. In this work, 3D-CCMs were used to evaluate the correlative efficacies of nanodrug exposure and medical radiation on cells contained in a tumor like sample. A comparison with monolayer cell cultures was performed showing the advantageous outcome and the higher potential of 3D-CCMs. This cheap and easy to handle approach is more ethical than in vivo experiments, thus, giving a fast evaluation of cellular responses to various treatments.

Mueller polarimetric imaging for fast macroscopic mapping of microscopic collagen matrix remodeling by smooth muscle cells.

Smooth muscle cells (SMCs) are critical players in cardiovascular disease development and undergo complex phenotype switching during disease progression. However, SMC phenotype is difficult to assess and track in co-culture studies. To determine the contractility of SMCs embedded within collagen hydrogels, we performed polarized light imaging and subsequent analysis based on Mueller matrices. Measurements were made both in the absence and presence of endothelial cells (ECs) in order to establish the impact of EC-SMC communication on SMC contractility. The results demonstrated that Mueller polarimetric imaging is indeed an appropriate tool for assessing SMC activity which significantly modifies the hydrogel retardance in the presence of ECs. These findings are consistent with the idea that EC-SMC communication promotes a more contractile SMC phenotype. More broadly, our findings suggest that Mueller polarimetry can be a useful tool for studies of spatial heterogeneities in hydrogel remodeling by SMCs.