Search

Collagen fibers play an important role in both the structure and function of various tissues in the human body. Visualization and quantitative measurements of collagen fibers are possible through imaging modalities such as second harmonic generation (SHG), but accurate segmentation of collagen fibers is difficult for datasets involving variable imaging depths due to the effects of scattering and absorption. Therefore, an objective approach to segmentation is needed for datasets with images of variable SHG intensity. In this study, a U‐Net convolutional neural network (CNN) was trained to accurately segment collagen‐positive pixels throughout SHG z‐stacks. CNN performance was benchmarked against other common thresholding techniques, and was found to outperform intensity‐based segmentation algorithms within an independent dataset, particularly at deeper imaging depths. These results indicate that a trained CNN can accurately segment collagen‐positive pixels within a wide range of imaging depths, which is useful for quantitative SHG imaging in thick tissues. [ABSTRACT FROM AUTHOR]

Collagen fiber organization requires characterization in many biomedical applications, but it is difficult to objectively quantify in standard histology tissue sections. Quantitative polarized light imaging is a low‐cost technique that allows for rapid measurement of collagen fiber orientation and thickness. In this study, we utilize a quantitative polarized light imaging system to characterize fiber orientation and thickness from wound sections. Full thickness skin wound sections that were previously stained with hematoxylin and eosin were used to assess collagen fiber content and organization at different points during the wound healing process. Overall, wounds exhibited a measurable increase in collagen fiber thickness and a nonlinear change in fiber reorganization within the wound. Our study demonstrates that quantitative polarized light imaging is an inexpensive alternative or supplement to standard histology protocols, requiring no additional stains or dyes, and yields repeatable quantitative assessments of collagen organization. [ABSTRACT FROM AUTHOR]

Soft-tissue regeneration methods currently yield suboptimal clinical outcomes due to loss of tissue volume and a lack of functional tissue regeneration. Grafted tissues and natural biomaterials often degrade or resorb too quickly, while most synthetic materials do not degrade. In previous research we demonstrated that soft-tissue regeneration can be supported using silk porous biomaterials for at least 18 months in vivo in a rodent model. In the present study, we scaled the system to a survival study using a large animal model and demonstrated the feasibility of these biomaterials for soft-tissue regeneration in adult horses. Both slow and rapidly degrading silk matrices were evaluated in subcutaneous pocket and intramuscular defect depots. We showed that we can effectively employ an equine model over 6 months to simultaneously evaluate many different implants, reducing the number of animals needed. Furthermore, we were able to tailor matrix degradation by varying the initial format of the implanted silk. Finally, we demonstrate ultrasound imaging of implants to be an effective means for tracking tissue regeneration and implant degradation. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1217-1227, 2015. [ABSTRACT FROM AUTHOR]

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker. [ABSTRACT FROM AUTHOR]

Background The disruption of neuron arrangement is associated with several pathologies. In contrast to action potentials, the role of resting potential (Vmem) in regulating connectivity remains unknown. Methods Neuron assemblies were quantified when their Vmem was depolarized using ivermectin (Ivm), a drug that opens chloride channels, for 24 h in cocultures with astrocytes. Cell aggregation was analyzed using automated cluster analysis methods. Neural connectivity was quantified based on the identification of isolated somas in phase-contrast images using image processing. Vmem was measured using voltage-sensitive dyes and whole-cell patch clamping. Immunocytochemistry and Western blotting were used to detect changes in the distribution and production of the proteins. Results Data show that Vmem regulates cortical tissue shape and connectivity. Automated cluster analysis methods revealed that the degree of neural aggregation was significantly increased (0.26 clustering factor vs. 0.21 in controls, P ≤ 0.01). The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm. Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures. Glia cell densities increased (33.3%, P ≤ 0.05) under depolarizing conditions. Conclusion Vmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements. [ABSTRACT FROM AUTHOR]

The goals of the present study are to establish an in vitro co-culture model of osteoblast and osteoclast function and to quantify the resulting bone remodeling. The bone is tissue engineered using well-defined silk protein biomaterials in 2D and 3D formats in combination with human cells. Parathyroid hormone (PTH) and glucose-dependent insulinotropic peptide (GIP) are selected because of their roles in bone remodeling for expression in tethered format on human mesenchymal stem cells (hMSCs). The cell-modified biomaterial surfaces are reconstructed from scanning electron microscopy images into 3D models for quantitative measurement of surface characteristics. Increased calcium deposition and surface roughness are found in 3D surface models of silk protein films remodeled by co-cultures containing tethered PTH, and decreased surface roughness is found for the films remodeled by tethered GIP co-cultures. Increased surface roughness is not found in monocultures of hMSCs expressing tethered PTH, suggesting that osteoclast-osteoblast interactions in the presence of PTH signaling are responsible for the increased mineralization. These data point towards the design of in vitro bone models in which osteoblast-osteoclast interactions are mimicked for a better understanding of bone remodeling. [ABSTRACT FROM AUTHOR]

Study Type – Therapy (case series) Level of Evidence 4 OBJECTIVE To describe various approaches for ureterolysis with an omental wrap using minimally invasive techniques, as surgery for idiopathic retroperitoneal fibrosis includes tissue biopsy, ureterolysis, and intraperitonealization or omental wrap. PATIENTS AND METHODS Since 2006 we have performed ureterolysis in four patients diagnosed with retroperitoneal fibrosis in two institutions. The ureterolysis in two cases was bilateral, using a standard laparoscopic approach for one case and a hand-assisted technique for the other. Unilateral ureterolysis was completed using a standard laparoscopic approach in one case and was converted to a hand-assisted technique in the other due to difficulty with ureteric identification. An omental wrap was used after ureterolysis for all renal units. RESULTS A minimally invasive technique was used for all ureterolysis procedures and none required open conversion. There was fascial dehiscence after surgery at the hand-port site in one patient, and required re-operation for wound closure. The median (range) hospital stay for all patients was 2.5 (2–10) days and the median blood loss was 100 (50–550) mL. No patient required a blood transfusion. At a median 16.5 (12–32) months of follow-up, there was symptomatic and radiographic success in all patients. CONCLUSIONS Ureterolysis can be a challenging operation depending on the extent of the retroperitoneal mass. An understanding of various laparoscopic techniques can provide the flexibility for successful completion of nearly all of these procedures using a minimally invasive approach. [ABSTRACT FROM AUTHOR]

Standard approaches to evaluate scar formation within histological sections rely on qualitative evaluations and scoring, which limits our understanding of the remodelling process. We have recently developed an image analysis technique for the rapid quantification of fibre alignment at each pixel location. The goal of this study was to evaluate its application for quantitatively mapping scar formation in histological sections of cutaneous burns. To this end, we utilized directional statistics to define maps of fibre density and directional variance from Masson's trichrome-stained sections for quantifying changes in collagen organization during scar remodelling. Significant increases in collagen fibre density are detectable soon after burn injury in a rat model. Decreased fibre directional variance in the scar was also detectable between 3 weeks and 6 months after injury, indicating increasing fibre alignment. This automated analysis of fibre organization can provide objective surrogate endpoints for evaluating cutaneous wound repair and regeneration. [ABSTRACT FROM AUTHOR]