The aim of this study was to determine the roles of collagen XII in the regulation of stromal hierarchical organization, keratocyte organization, and corneal mechanics. METHODS. The temporal and spatial expression of collagen XII at postnatal days 4, 10, 30, 90, and 150 were evaluated in wild-type (WT) mice. The role of collagen XII in hierarchical organization was analyzed by measuring fibril diameter and density, as well as stromal lamellar structure, within ultrastructural micrographs obtained from WT and collagen XIIdeficient mice (Col12a1-/-). Keratocyte morphology and networks were assessed using actin staining with phalloidin and in vivo confocal microscopy. The effects of collagen XII on corneal biomechanics were evaluated with atomic force microscopy. RESULTS. Collagen XII was localized homogeneously in the stroma from postnatal day 4 to day 150, and protein accumulation was shown to increase during this period using semiquantitative immunoblots. Higher fibril density (P < 0.001) and disruption of lamellar organization were found in the collagen XII null mice stroma when compared to WT mice. Keratocyte networks and organization were altered in the absence of collagen XII, as demonstrated using fluorescent microscopy after phalloidin staining and in vivo confocal microscopy. Corneal stiffness was increased in the absence of collagen XII. Young's modulus was 16.2 ± 5.6 kPa in WT and 32.8 ± 6.4 kPa in Col12a1-/corneas. The difference between these two groups was significant (P < 0.001, t-test). CONCLUSIONS. Collagen XII plays a major role in establishing and maintaining stromal structure and function. In the absence of collagen XII, the corneal stroma showed significant abnormalities, including decreased interfibrillar space, disrupted lamellar organization, abnormal keratocyte organization, and increased corneal stiffness.

Objective: To date, the pathophysiology of the meniscus has not been fully elucidated. Due to the tissue's limited vascularization, nutrients and other molecular signals spread through the extracellular matrix via diffusion or convection (interstitial fluid flow). Understanding transport mechanisms is crucial to elucidating meniscal pathophysiology, and to designing treatments for repair and restoration of the tissue. Similar to other fibrocartilaginous structures, meniscal morphology and composition may affect its diffusive properties. The objective of this study was to investigate the role of solute size, and tissue structure and composition on molecular diffusion in meniscus tissue. Design: Using a custom FRAP technique developed in our lab, we measured the direction-dependent diffusivity in human meniscus of six different molecular probes of size ranging from~300Da to 150,000Da. Diffusivity measurements were related to sample water content. SEM images were used to investigate collagen structure in relation to transport mechanisms. Results: Diffusivity was anisotropic, being significantly faster in the direction parallel to collagen fibers when compared the orthogonal direction. This was likely due to the unique structural organization of the tissue presenting pores aligned with the fibers, as observed in SEM images. Diffusion coefficients decreased as the molecular size increased, following the Ogston model. No significant correlations were found among diffusion coefficients and water content of the tissue. Conclusions: This study provides new knowledge on the mechanisms of molecular transport in meniscal tissue. The reported results can be leveraged to further investigate tissue pathophysiology and to design treatments for tissue restoration or replacement.

No abstract

Pancreatic islet transplantation improves metabolic control and prevents complications in patients with brittle type 1 diabetes (T1D). However, chronic immunosuppression is required to prevent allograft rejection and recurrence of autoimmunity. Islet encapsulation may eliminate the need for immunosuppression. Here, we analyzed in parallel two microencapsulation platforms that provided long-term diabetes reversal in preclinical T1D models, alginate single and double capsules versus polyethylene glycol conformal coating, to identify benefits and weaknesses that could inform the design of future clinical trials with microencapsulated islets. We performed in vitro and in vivo functionality assays with human islets and analyzed the explanted grafts by immunofluorescence. We quantified the size of islets and capsules, measured capsule permeability, and used these data for in silico simulations of islet functionality in COMSOL Multiphysics. We demonstrated that insulin response to glucose stimulation is dependent on capsule size, and the presence of permselective materials augments delays in insulin secretion. Non-coated and conformally coated islets could be transplanted into the fat pad of diabetic mice, resulting in comparable functionality and metabolic control. Mac-2+ cells were found in conformally coated grafts, indicating possible host reactivity. Due to their larger volume, alginate capsules were transplanted in the peritoneal cavity. Despite achieving diabetes reversal, changes in islet composition were found in retrieved capsules, and recipient mice experienced hypoglycemia indicative of hyperinsulinemia induced by glucose retention in large capsules as the in silico model predicted. We concluded that minimal capsule size is critical for physiological insulin secretion, and anti-inflammatory modulation may be beneficial for small conformal capsules.