Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

BackgroundArticular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries) as our model animal.ResultsPredominant collagen orientation is parallel to the articular surface throughout the tissue depth for perinatal cartilage. This remodels to the Benninghoff structure before the sheep reach sexual maturity. Remodelling of predominant collagen orientation starts at a depth just below the future transitional zone. Tissue retardance shows a minimum near the articular surface at all ages, which indicates the presence of zonal differentiation at all ages. The absolute position of this minimum does change between birth and maturity. Between different anatomical sites, we find differences in the dynamics of collagen remodelling, but no differences in adult collagen structure.ConclusionsThe collagen network in articular cartilage remodels between birth and sexual maturity from a network with predominant orientation parallel to the articular surface to a Benninghoff network. The retardance minimum near, but not at, the articular surface at all ages shows that a zonal differentiation is already present in the perinatal animals. In these animals, the zonal differentiation can not be correlated to the collagen network orientation. We find no difference in adult collagen structure in the nearly congruent metacarpophalangeal joint, but we do find differences in the dynamics of collagen network remodelling.

Summary In the presence of anisotropic biochemical or topographical patterns, cells tend to align in the direction of these cues—a widely reported phenomenon known as “contact guidance.” To investigate the origins of contact guidance, here, we created substrates micropatterned with parallel lines of fibronectin with dimensions spanning multiple orders of magnitude. Quantitative morphometric analysis of our experimental data reveals two regimes of contact guidance governed by the length scale of the cues that cannot be explained by enforced alignment of focal adhesions. Adopting computational simulations of cell remodeling on inhomogeneous substrates based on a statistical mechanics framework for living cells, we show that contact guidance emerges from anisotropic cell shape fluctuation and “gap avoidance,” i.e., the energetic penalty of cell adhesions on non-adhesive gaps. Our findings therefore point to general biophysical mechanisms underlying cellular contact guidance, without the necessity of invoking specific molecular pathways.

By injecting a few cubic centimeters of saline into the coronary artery and using thermodilution principles, mean transit time (T mn ) of the injectate can be calculated and is inversely proportional to coronary blood flow. Because microvascular resistance equals distal coronary pressure (P d ) divided by myocardial flow, the product P d ⅐ T mn provides an index of myocardial resistance (IMR). In this in vitro study in a physiologic model of the coronary circulation, we compared IMR to true myocardial resistance (TMR) at different degrees of myocardial resistance and at different degrees of epicardial stenosis. Absolute blood flow was varied from 42 to 203 ml/min and TMR varied from 0.39 to 1.63 dynes ⅐ sec/cm 5 . Inverse mean transit time correlated well to absolute blood flow (R 2 ‫؍‬ 0.93). Furthermore, an excellent correlation was found between IMR and TMR (R 2 ‫؍‬ 0.94). IMR was independent on the severity of epicardial stenosis and thus specific for myocardial resistance. Thus, using one single guidewire, both fractional flow reserve and IMR can be measured simultaneously as indexes of epicardial and microvascular disease, respectively, enabling separate assessment of both coronary arterial and microvascular disease.

Quantitative polarized light microscopy (qPLM) is a popular tool for the investigation of birefringent architectures in biological tissues. Collagen, the most abundant protein in mammals, is such a birefringent material. Interpretation of results of qPLM in terms of collagen network architecture and anisotropy is challenging, because different collagen networks may yield equal qPLM results. We created a model and used the linear optical behavior of collagen to construct a Jones or Mueller matrix for a histological cartilage section in an optical qPLM train. Histological sections of tendon were used to validate the basic assumption of the model. Results show that information on collagen densities is needed for the interpretation of qPLM results in terms of collagen anisotropy. A parameter that is independent of the optical system and that measures collagen fiber anisotropy is introduced, and its physical interpretation is discussed. With our results, we can quantify which part of different qPLM results is due to differences in collagen densities and which part is due to changes in the collagen network. Because collagen fiber orientation and anisotropy are important for tissue function, these results can improve the biological and medical relevance of qPLM results.