Microneurotrophin BNN27 Reduces Astrogliosis and Increases Density of Neurons and Implanted Neural Stem Cell-Derived Cells after Spinal Cord Injury.

Microneurotrophins, small-molecule mimetics of endogenous neurotrophins, have demonstrated significant therapeutic effects on various animal models of neurological diseases. Nevertheless, their effects on central nervous system injuries remain unknown. Herein, we evaluate the effects of microneurotrophin BNN27, an NGF analog, in the mouse dorsal column crush spinal cord injury (SCI) model. BNN27 was delivered systemically either by itself or combined with neural stem cell (NSC)-seeded collagen-based scaffold grafts, demonstrated recently to improve locomotion performance in the same SCI model. Data validate the ability of NSC-seeded grafts to enhance locomotion recovery, neuronal cell integration with surrounding tissues, axonal elongation and angiogenesis. Our findings also show that systemic administration of BNN27 significantly reduced astrogliosis and increased neuron density in mice SCI lesion sites at 12 weeks post injury. Furthermore, when BNN27 administration was combined with NSC-seeded PCS grafts, BNN27 increased the density of survived implanted NSC-derived cells, possibly addressing a major challenge of NSC-based SCI treatments. In conclusion, this study provides evidence that small-molecule mimetics of endogenous neurotrophins can contribute to effective combinatorial treatments for SCI, by simultaneously regulating key events of SCI and supporting grafted cell therapies in the lesion site.

Mammals fail to regenerate organs when wound contraction drives scar formation.

To understand why mammals generally do not regenerate injured organs, we considered the exceptional case of spontaneous skin regeneration in the early lamb fetus. Whereas during the early fetal stage skin wounds heal by regeneration, in the late fetal stage, and after birth, skin wounds close instead by scar formation. We review independent evidence that this switch in wound healing response coincides with the onset of wound contraction, which is also enabled during late fetal gestation. The crucial role of wound contraction in determining the wound healing outcome in adults has been demonstrated in three mammalian models of severe injury (excised guinea pig skin, transected rat sciatic nerve, excised rabbit conjunctival stroma) where grafting the injury with DRT, a contraction-blocking scaffold of highly-specific structure, altered significantly the wound healing outcome. While spontaneous healing resulted in scar formation in these animal models, DRT grafting significantly reduced the extent of wound contraction, prevented scar synthesis, and resulted in partial regeneration. These findings, as well as independent data from species that heal spontaneously via regeneration, point to a striking hypothesis: The process of regeneration lies dormant in mammals until appropriately activated by injury. In spontaneous wound healing of the late fetus and in adult mammals, wound contraction impedes such endogenous regeneration mechanisms. However, engineered treatments, such as DRT, that block wound contraction can cancel its effects and favor wound healing by regeneration instead of scar formation.

Three-dimensional characterization of collagen remodeling in cell-seeded collagen scaffolds via polarization second harmonic generation.

In this study, we use non-linear imaging microscopy to characterize the structural properties of porous collagen-GAG scaffolds (CGS) seeded with human umbilical vein endothelial cells (HUVECs), as well as human mesenchymal stem cells (hMSCs), a co-culture previously reported to form vessel-like structures inside CGS. The evolution of the resulting tissue construct was monitored over 10 days via simultaneous two- and three-photon excited fluorescence microscopy. Time-lapsed 2- and 3-photon excited fluorescence imaging was utilized to monitor the temporal evolution of the vascular-like structures up to 100 µm inside the scaffold up to 10 days post-seeding. 3D polarization-dependent second harmonic generation (PSHG) was utilized to monitor collagen-based scaffold remodeling and determine collagen fibril orientation up to 200 µm inside the scaffold. We demonstrate that polarization-dependent second harmonic generation can provide a novel way to quantify the reorganization of the collagen architecture in CGS simultaneously with key biomechanical interactions between seeded cells and CGS that regulate the formation of vessel-like structures inside 3D tissue constructs. A comparison between samples at different days in vitro revealed that gradually, the scaffolds developed an orthogonal net-like architecture, previously found in real skin.

Neural stem cell delivery via porous collagen scaffolds promotes neuronal differentiation and locomotion recovery in spinal cord injury.

Neural stem cell (NSC) grafts have demonstrated significant effects in animal models of spinal cord injury (SCI), yet their clinical translation remains challenging. Significant evidence suggests that the supporting matrix of NSC grafts has a crucial role in regulating NSC effects. Here we demonstrate that grafts based on porous collagen-based scaffolds (PCSs), similar to biomaterials utilized clinically in induced regeneration, can deliver and protect embryonic NSCs at SCI sites, leading to significant improvement in locomotion recovery in an experimental mouse SCI model, so that 12 weeks post-injury locomotion performance of implanted animals does not statistically differ from that of uninjured control animals. NSC-seeded PCS grafts can modulate key processes required to induce regeneration in SCI lesions including enhancing NSC neuronal differentiation and functional integration in vivo, enabling robust axonal elongation, and reducing astrogliosis. Our findings suggest that the efficacy and translational potential of emerging NSC-based SCI therapies could be enhanced by delivering NSC via scaffolds derived from well-characterized clinically proven PCS.

Regeneration mechanism for skin and peripheral nerves clarified at the organ and molecular scales.

This article is a review of current research on the mechanism of regeneration of skin and peripheral nerves based on use of collagen scaffolds, particularly the dermis regeneration template (DRT), which is widely used clinically. DRT modifies the normal wound healing process, converting it from wound closure by contraction and scar formation to closure by regeneration. DRT achieves this modification by blocking wound contraction, which spontaneously leads to cancellation of scar formation, a process secondary to contraction. Contraction blocking by DRT is the result of a dramatic phenotype change in contractile cells (myofibroblasts, MFB) which follows specific binding of integrins α1ß1 and α2ß1 onto hexapeptide ligands, probably GFOGER and GLOGER, that are naturally present on the surface of collagen fibers in DRT. The methodology of organ regeneration based on use of DRT has been recently extended from traumatized skin to diseased skin. Successful extension of the method to other organs in which wounds heal by contraction is highly likely though not yet attempted. This regenerative paradigm is much more advanced both in basic mechanistic understanding and clinical use than methods based on tissue culture or stem cells. It is also largely free of risk and has shown decisively lower morbidity and lower cost than organ transplantation.

Regeneration of injured skin and peripheral nerves requires control of wound contraction, not scar formation.

We review the mounting evidence that regeneration is induced in wounds in skin and peripheral nerves by a simple modification of the wound healing process. Here, the process of induced regeneration is compared to the other two well-known processes by which wounds close, i.e., contraction and scar formation. Direct evidence supports the hypothesis that the mechanical force of contraction (planar in skin wounds, circumferential in nerve wounds) is the driver guiding the orientation of assemblies of myofibroblasts (MFB) and collagen fibers during scar formation in untreated wounds. We conclude that scar formation depends critically on wound contraction and is, therefore, a healing process secondary to contraction. Wound contraction and regeneration did not coincide during healing in a number of experimental models of spontaneous (untreated) regeneration described in the literature. Furthermore, in other studies in which an efficient contraction-blocker, a collagen scaffold named dermis regeneration template (DRT), and variants of it, were grafted on skin wounds or peripheral nerve wounds, regeneration was systematically observed in the absence of contraction. We conclude that contraction and regeneration are mutually antagonistic processes. A dramatic change in the phenotype of MFB was observed when the contraction-blocking scaffold DRT was used to treat wounds in skin and peripheral nerves. The phenotype change was directly observed as drastic reduction in MFB density, dispersion of MFB assemblies and loss of alignment of the long MFB axes. These observations were explained by the evidence of a surface-biological interaction of MFB with the scaffold, specifically involving binding of MFB integrins α1 ß1 and α2 ß1 to ligands GFOGER and GLOGER naturally present on the surface of the collagen scaffold. In summary, we show that regeneration of wounded skin and peripheral nerves in the adult mammal can be induced simply by appropriate control of wound contraction, rather than of scar formation.

In Situ Quantification of Surface Chemistry in Porous Collagen Biomaterials.

Cells inside a 3D matrix (such as tissue extracellular matrix or biomaterials) sense their insoluble environment through specific binding interactions between their adhesion receptors and ligands present on the matrix surface. Despite the critical role of the insoluble matrix in cell regulation, there exist no widely-applicable methods for quantifying the chemical stimuli provided by a matrix to cells. Here, we describe a general-purpose technique for quantifying in situ the density of ligands for specific cell adhesion receptors of interest on the surface of a 3D matrix. This paper improves significantly the accuracy of the procedure introduced in a previous publication by detailed marker characterization, optimized staining, and improved data interpretation. The optimized methodology is utilized to quantify the ligands of integrins α 1 ß 1, α 2 ß 1 on two kinds of matched porous collagen scaffolds, which are shown to possess significantly different ligand density, and significantly different ability to induce peripheral nerve regeneration in vivo. Data support the hypothesis that cell adhesion regulates contractile cell phenotypes, recently shown to be inversely related to organ regeneration. The technique provides a standardized way to quantify the surface chemistry of 3D matrices, and a means for introducing matrix effects in quantitative biological models.

Spectral-resolved multifocal multiphoton microscopy with multianode photomultiplier tubes.

Multiphoton excitation fluorescence microscopy is the preferred method for in vivo deep tissue imaging. Many biological applications demand both high imaging speed and the ability to resolve multiple fluorophores. One of the successful methods to improve imaging speed in a highly turbid specimen is multifocal multiphoton microscopy (MMM) based on use of multi-anode photomultiplier tubes (MAPMT). This approach improves imaging speed by using multiple foci for parallelized excitation without sacrificing signal to noise ratio (SNR) due to the scattering of emission photons. In this work, we demonstrate that the MAPMT based MMM can be extended with spectral resolved imaging capability. Instead of generating multiple excitation foci in a 2D grid pattern, a linear array of foci is generated. This leaves one axis of the 2D MAPMT available for spectral dispersion and detection. The spectral-resolved MMM can detect several emission signals simultaneously with high imaging speed optimized for high-throughput, high-contents applications. The new procedure is illustrated using imaging data from the kidney, peripheral nerve regeneration and dendritic morphological data from the brain.

3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation.

Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude.

Common features of optimal collagen scaffolds that disrupt wound contraction and enhance regeneration both in peripheral nerves and in skin.

The adult mammal responds to severe injury of most organs spontaneously by wound contraction and scar formation, rather than by regeneration. In severe skin wounds, the ability of porous collagen scaffolds to induce regeneration was found to correlate strongly with a reduction in wound contraction rate. Here, we present quantitative evidence of a similar positive relationship between the extent of disruption of tissue contraction and quality of peripheral nerve regeneration in transected rat peripheral nerves. Our observations suggest that porous collagen scaffolds enhance regeneration both in injured adult skin and peripheral nerves by disrupting the formation of a contractile cell capsule at the edges of the wound. Preliminary observations made with other injured organs support the hypothesis that capsules or clusters of contractile cells impose a universal mechanical barrier during wound healing which, if disrupted appropriately, enhances the quality of induced regeneration in a wider range of organs.

Quantifying the dynamics of bacterial secondary metabolites by spectral multiphoton microscopy.

Phenazines, a group of fluorescent small molecules produced by the bacterium Pseudomonas aeruginosa, play a role in maintaining cellular redox homeostasis. Phenazines have been challenging to study in vivo due to their redox activity, presence both intra- and extracellularly, and their diverse chemical properties. Here, we describe a noninvasive in vivo optical technique to monitor phenazine concentrations within bacterial cells using time-lapsed spectral multiphoton fluorescence microscopy. This technique enables simultaneous monitoring of multiple weakly fluorescent molecules (phenazines, siderophores, NAD(P)H) expressed by bacteria in culture. This work provides the first in vivo measurements of reduced phenazine concentration as well as the first description of the temporal dynamics of the phenazine-NAD(P)H redox system in Pseudomonas aeruginosa, illuminating an unanticipated role for 1-hydroxyphenazine. Similar approaches could be used to study the abundance and redox dynamics of a wide range of small molecules within bacteria, both as single cells and in communities.

An optical method to quantify the density of ligands for cell adhesion receptors in three-dimensional matrices.

The three-dimensional matrix that surrounds cells is an important insoluble regulator of cell phenotypes. Examples of such insoluble surfaces are the extracellular matrix (ECM), ECM analogues and synthetic polymeric biomaterials. Cell-matrix interactions are mediated by cell adhesion receptors that bind to chemical entities (adhesion ligands) on the surface of the matrix. There are currently no established methods to obtain quantitative estimates of the density of adhesion ligands recognized by specific cell adhesion receptors. This article presents a new optical-based methodology for measuring ligands of adhesion receptors on three-dimensional matrices. The study also provides preliminary quantitative results for the density of adhesion ligands of integrins alpha(1)beta(1) and alpha(2)beta(1) on the surface of collagen-based scaffolds, similar to biomaterials that are used clinically to induce regeneration in injured skin and peripheral nerves. Preliminary estimates of the surface density of the ligands of these two major collagen-binding receptors are 5775 +/- 2064 ligands microm(-2) for ligands of alpha(1)beta(1) and 17 084 +/- 5353 ligands microm(-2) for ligands of alpha(2)beta(1). The proposed methodology can be used to quantify the surface chemistry of insoluble surfaces that possess biological activity, such as native tissue ECM and biomaterials, and therefore can be used in cell biology, biomaterials science and regenerative medical studies for quantitative description of a matrix and its effects on cells.

Changes in the biochemical constituents and morphologic appearance of the human cervical stroma during pregnancy.

OBJECTIVE: The cervix is the lower portion of the uterus. It is composed of fibrous tissue and its mechanical integrity is crucial for maintaining a healthy gestation. During normal pregnancy, the cervical extracellular matrix (ECM) remodels in preparation for labor. The objective of this study was to investigate the biochemical and morphological changes in cervical stroma associated with physiological remodeling during normal pregnancy. STUDY DESIGN: Using human cervical tissue obtained from pregnant and non-pregnant patients, the ECM was analyzed for its biochemical constituents and histologic morphology. The ECM was assayed for hydration, collagen concentration, collagen solubility, total sulfated glycosaminoglycan concentration, and individual disaccharide concentration. The ECM morphology was visualized using conventional histological techniques (Masson's trichrome stain, polarized light microscopy) as well as second harmonic generation (SHG) imaging. RESULTS: When comparing pregnant to non-pregnant tissue, significant increases were measured for total sulfated glycosaminoglycans, hyaluronic acid, and collagen solubility. The microscopy studies confirmed that the collagenous network of the cervical stroma was anisotropic and pregnancy was associated with a discernable decrease in collagen organization. CONCLUSION: Significant changes were seen in the concentration and organization of cervical ECM constituents during normal pregnancy.