Medial collagen organization in human arteries of the heart and brain by polarized light microscopy.

The mechanical properties of collagen as a biopolymer ensures that collagen has a significant influence on the mechanical behavior of the host tissue. Structural organization is a key to that influence. We have assessed this relationship quantitatively in the tunica media of arteries from the heart and brain, using the polarizing light microscope and Universal stage. Arteries from 22 autopsies were isolated, cannulated and fixed with 10% buffered formalin, at a distending pressure spanning normal values in vivo. We prepared the tissue for light microscopy, with paraffin embedding, sectioning at 7 microns, and staining with picrosirius red to enhance the natural birefringence of medial collagen. Individual measurements, 30 to 50 per arterial section, referenced against the central axis of the vessel segment, revealed a coherent organization, with an average orientation which was within 1 to 2 degrees of being perfectly concentric for all artery segments. Analysis was done with Lambert projections and circular statistics. We calculated the circular standard deviation, which was 5.2 degrees for 27 brain arteries (S.D. 1.9 degrees) and 5.6 degrees (S.D. 2.1 degrees), for 5 coronary arteries sectioned at less than 15 degrees. Our interpretation is that medial collagen can be strained even though highly aligned, revealing a mechanical property which contrasts that of type I collagen.     

Effect of Decorin and Dermatan Sulfate on the Mechanical Properties of a Neocartilage

Decorin is known to influence the size of collagen fibrils in ligaments and tendons and it has been hypothesized to provide a structural link between collagen fibrils in connective tissues, including cartilage. Coincidently, mechanical properties of skin, ligament, and tendons are altered in decorin knockout mice, suggesting it may influence the structural properties of tissue or tissue matrix organization. To further examine the role of decorin in the extracellular matrix development and subsequent material properties of cartilage, tissue (neocartilage) was grown in a 3D culture model using a pure population of genetically modified chondrocytes stably overexpressing decorin (DCN) or decorin lacking dermatan sulfate (MDCN). An empty vector (CON) served as a virus control. Following generation of the cartilage-like tissues, mechanical properties in tension and compression, collagen fibril diameter, matrix organization, and biochemistry of the tissue were determined. There were no differences between CON and DCN tissues in any parameter measured. In contrast, tissue generated in MDCN cultures was thinner, had higher collagen density, and higher elastic moduli as compared to both CON and DCN tissues. Considering there was no difference in stiffness between CON and DCN tissues, the notion that decorin contributes to the mechanical properties via load transfer was refuted in this model. However, contrasts in the mechanical properties of the MDCN tissue suggest that the dermatan sulfate chains on decorin influences the organization/maturation and resultant mechanical properties of the matrix by as an yet-unidentified regulatory mechanism.     

Mechanical Properties and Compositions of Tissue Engineered and Native Arteries

With the goal of mimicking the mechanical properties of a given native tissue, tissue engineers seek to culture replacement tissues with compositions similar to those of native tissues. In this report, differences between the mechanical properties of engineered arteries and native arteries were correlated with differences in tissue composition. Engineered arteries failed to match the strengths or compliances of native tissues. Lower strengths of engineered arteries resulted partially from inferior organization of collagen, but not from differences in collagen density. Furthermore, ultimate strengths of engineered vessels were significantly reduced by the presence of residual polyglycolic acid polymer fragments, which caused stress concentrations in the vessel wall. Lower compliances of engineered vessels resulted from minimal smooth muscle cell contractility and a lack of organized extracellular elastin. Organization of elastin and collagen in engineered arteries may have been partially hindered by high concentrations of sulfated glycosaminoglycans. Tissue engineers should continue to regulate cell phenotype and promote synthesis of proteins that are known to dominate the mechanical properties of the associated native tissue. However, we should also be aware of the potential negative impacts of polymer fragments and glycosaminoglycans on the mechanical properties of engineered tissues.     

The molecular organization of collagen in saccular aneurysms assessed by polarized light microscopy

The known relationship between the retardation of polarized light and the molecular organization of collagen enabled us to study collagen in cerebral saccular aneurysms, structures in which the collagen is probably abnormal. Six aneurysms and their adjacent arteries, obtained at autopsy, were sectioned at a thickness of 7 microns. Measurements of retardation in hematoxylin and eosin stained sections, obtained by the method of de Sénarmont, were significantly lower in the aneurysm wall compared with the adventitial region of adjacent arteries. A similar reduction in retardation was found by imbibition analysis on unstained sections from three of the aneurysms. We propose that the difference in optical properties arises because the aneurysm collagen is immature and so is less organized at the molecular level. The presence of immature collagen could also explain the altered mechanical properties that are necessary for the enlargement and rupture of saccular aneurysms.     

Birefringence and Second Harmonic Generation on Tendon Collagen Following Red Linearly Polarized Laser Irradiation

Regarding the importance of type I collagen in understanding the mechanical properties of a range of tissues, there is still a gap in our knowledge of how proteins perform such work. There is consensus in literature that the mechanical characteristics of a tissue are primarily determined by the organization of its molecules. The purpose of this study was to characterize the organization of non-irradiated and irradiated type I collagen. Irradiation was performed with a linearly polarized HeNe laser (λ = 632.8 nm) and characterization was undertaken using polarized light microscopy to investigate the birefringence and second harmonic generation to analyze nonlinear susceptibility. Rats received laser irradiation (P = 6.0 mW, I = 21.2 mW/cm², E ≈ 0.3 J, ED = 1.0 J/cm²) on their healthy Achilles tendons, which after were extracted to prepare the specimens. Our results show that irradiated samples present higher birefringence and greater non-linear susceptibility than non-irradiated samples. Under studied conditions, we propose that a red laser with polarization direction aligned in parallel to the tendon long axis promotes further alignment on the ordered healthy collagen fibrils towards the electric field incident. Thus, prospects for biomedical applications for laser polarized radiation on type I collagen are encouraging since it supports greater tissue organization.     

Layered collagen fabric of cerebral aneurysms quantitatively assessed by the universal stage and polarized light microscopy.

We evaluated the effectiveness of the Universal stage, an instrument for measuring three-dimensional orientation of birefringent materials, for studying the collagen fabric in the wall of brain aneurysms. Vessels from autopsy were fixed at normal arterial distending pressure with 10% formalin, and prepared for polarized light microscopy, with paraffin embedding and staining with picrosirius red for birefringent enhancement. Quantitative data were obtained from tangential and oblique sections (7 microns thickness) of an intact 8 mm aneurysm, a 1.5 mm aneurysm, and a tangential section (3 microns thickness) of a cerebral artery. Sections of full-size aneurysms seen through the microscope, adjusted either for plane or circularly polarized light, revealed distinctive layers of collagen across the aneurysmal wall, which at higher magnification were further subdivided. Three-dimensional measurements, numbering 1,082, were made by use of the Universal stage attachment to the polarizing microscope. They were plotted by computer-controlled graphics on Lambert projections and analyzed by circular statistics. When assessed layer by layer, the collagen spanned a full range of orientations relative to the tangential plane. The circular standard deviation, a measure of the spread of alignment about the mean, was as low as 10 degrees for coherently organized collagen and as high as 40 degrees for the least coherently organized collagen, values characteristic of either the organized tunica media, or the least organized tunica adventitia of cerebral arteries. Although there was a marked thinning of the wall of one aneurysm, there was no evidence of structural weakness based only on the directional organization assessed by our measurements.     

Quantification of the Temporal Evolution of Collagen Orientation in Mechanically Conditioned Engineered Cardiovascular Tissues

Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network. The work was performed at the Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.     

Fabrication of branched hybrid vascular prostheses

We devised a branched, or bifurcated, hybrid vascular prosthesis that was mainly composed of bovine smooth muscle cells (SMCs) and type I collagen with minimal reinforcement by a knitted fabric mesh made of segmented polyester. The tubular hybrid medial tissue with small (3 mm) or large (6 mm) inner diameter was prepared by pouring a cold mixed solution of SMCs and collagen into a corresponding tubular mold and by subsequent thermal gelation, followed by 7-day culturing. A branched hybrid medial tissue was prepared by an end-to-side anastomosis between these tubes of different sizes. Two-week culture of the branched tissue resulted in continuous tissue formation at the anastomotic site. Upon seeding and culture of bovine endothelial cells (ECs), a fully endothelialized branched hybrid vessel was prepared. Under a continuous pulsatile flow condition, morphological alterations of ECs, responding to local flow dynamics generated by the branched configuration, were seen. Reinforcement with an elastomeric mesh improved mechanical strength of the hybrid tissue and created compliance matching with native arteries. A branched hybrid graft with mesh reinforcement is expected to be applicable to arterial replacement in a branching region.     

Intermittent straining accelerates the development of tissue properties in engineered heart valve tissue

Tissue-engineered heart valves lack sufficient amounts of functionally organized structures and consequently do not meet in vivo mechanical demands. To optimize tissue architecture and hence improve mechanical properties, various in vitro mechanical conditioning protocols have been proposed, of which intermittent straining is most promising in terms of tissue properties. We hypothesize that this is due to an improved collagen matrix synthesis, maturation, and organization, triggered by periodic straining of cells. To test this hypothesis, we studied the effect of intermittent versus constrained conditioning with time (2-4 weeks), using a novel model system of human heart valve tissue. Temporal variations in collagen production, cross-link density, and mechanical properties were quantified in engineered heart valve tissue, cyclically strained for 3-h periods, alternated with 3-h periods rest. In addition, an innovative method for vital collagen imaging was used to monitor collagen organization. Intermittent straining resulted in increased collagen production, cross-link densities, collagen organization, and mechanical properties at faster rates, as compared to constrained controls, leading to stronger tissues in shorter culture periods. This is of utmost importance for heart valve tissue engineering, where insufficient mechanical properties are currently the main limiting factor.     

Vascular tissue engineering: microtextured scaffold templates to control organization of vascular smooth muscle cells and extracellular matrix

The in vitro construction of tissue-engineered small diameter (<6mm) blood vessels with sufficient strength and mechanical compliance has evaded researchers. We hypothesize that the high spatial organization of the medial layer of vascular smooth muscle cells (VSMCs) and their surrounding matrix provides high burst strength, compliance, and stability. We investigated the effect of microfabricated polydimethylsiloxane (PDMS) scaffolds with various groove widths on VSMC organization. We found that the presence of these grooved topographical cues significantly enhanced VSMC aspect ratio, alignment, and oriented remodeling of the underlying extracellular matrix. This study suggests that topographical patterning of tissue scaffolds can influence cellular and matrix spatial organization and could provide a framework for achieving the required organization and physical properties for blood vessels.     

Abnormal collagen fibrils in aspartylglycosaminuria. Altered dermal ultrastructure in a glycoprotein storage disorder

Patients with aspartylglycosaminuria, a lysosomal storage disorder of glycoprotein degradation, express connective tissue signs that refer to impaired mechanical properties of the tissue. We studied the ultrastructure of the dermis of patients with aspartylglycosaminuria to detect possible alterations in the connective tissue matrix, alterations that could explain the clinical findings. The organization of fiber bundles was studied by light microscopy and scanning electron microscopy, and diameters and volume densities of individual collagen fibrils were measured. The histologic organization of the dermis in patients with aspartylglycosaminuria was normal. However, by scanning electron microscopy a looser organization and more irregular orientation of the fiber bundles were detected. Transmission electron microscopy revealed a strikingly abnormal variation in the diameters of individual collagen fibrils (from 20 to 160 nm) in all layers of the dermis, with slight irregularity of shape especially in the thickest fibrils. Occasional giant fibrils (greater than 200 nm) were observed. The distribution of the ruthenium red-positive material around the fibrils was normal. Ultrastructural changes similar to these have been found in the collagen fibrils of some patients with Ehlers-Danlos syndrome as well as certain other disorders affecting dermal connective tissue. Altered collagen fibril formation offers an explanation for the connective tissue lesions in aspartylglycosaminuria.     

Altered structural and mechanical properties in decellularized rabbit carotid arteries

Recently, major achievements in creating decellularized whole tissue scaffolds have drawn considerable attention to decellularization as a promising approach for tissue engineering. Decellularized tissues are expected to have mechanical strength and structure similar to the native tissues from which they are derived. However, numerous studies have shown that mechanical properties change after decellularization. Often, tissue structure is observed by histology and electron microscopy, but the structural alterations that may have occurred are not always evident. Here, a variety of techniques were used to investigate changes in tissue structure and relate them to altered mechanical behavior in decellularized rabbit carotid arteries. Histology and scanning electron microscopy revealed that major extracellular matrix components were preserved and fibers appeared intact, although collagen appeared looser and less crimped after decellularization. Transmission electron microscopy confirmed the presence of proteoglycans (PG), but there was decreased PG density and increased spacing between collagen fibrils. Mechanical testing and opening angle measurements showed that decellularized arteries had significantly increased stiffness, decreased extensibility and decreased residual stress compared with native arteries. Small-angle light scattering revealed that fibers had increased mobility and that structural integrity was compromised in decellularized arteries. Taken together, these studies revealed structural alterations that could be related to changes in mechanical properties. Further studies are warranted to determine the specific effects of different decellularization methods on the structure and performance of decellularized arteries used as vascular grafts.     

Tendon and ligament fibrillar crimps give rise to left-handed helices of collagen fibrils in both planar and helical crimps

Collagen fibres in tendons and ligaments run straight but in some regions they show crimps which disappear or appear more flattened during the initial elongation of tissues. Each crimp is formed of collagen fibrils showing knots or fibrillar crimps at the crimp top angle. The present study analyzes by polarized light microscopy, scanning electron microscopy, transmission electron microscopy the 3D morphology of fibrillar crimp in tendons and ligaments of rat demonstrating that each fibril in the fibrillar region always twists leftwards changing the plane of running and sharply bends modifying the course on a new plane. The morphology of fibrillar crimp in stretched tendons fulfills the mechanical role of the fibrillar crimp acting as a particular knot/biological hinge in absorbing tension forces during fibril strengthening and recoiling collagen fibres when stretching is removed. The left‐handed path of fibrils in the fibrillar crimp region gives rise to left‐handed fibril helices observed both in isolated fibrils and sections of different tendons and ligaments (flexor digitorum profundus muscle tendon, Achilles tendon, tail tendon, patellar ligament and medial collateral ligament of the knee). The left‐handed path of fibrils represents a new final suprafibrillar level of the alternating handedness which was previously described only from the molecular to the microfibrillar level. When the width of the twisting angle in the fibrillar crimp is nearly 180° the fibrils appear as left‐handed flattened helices forming crimped collagen fibres previously described as planar crimps. When fibrils twist with different subsequent rotational angles (< 180°) they always assume a left‐helical course but, running in many different nonplanar planes, they form wider helical crimped fibres.     

Collagen Gel Anisotropy Measured by 2-D Laser Trap Microrheometry

Collagen gels can serve as biomaterials ideal for tissue equivalents, especially if they are remodeled to have fibril anisotropy mimicking native tissue. Type I collagen gel remodeling was studied microscopically to investigate the changes caused by fibroblasts in collagen gel structures, with and without the growth factors PDGF-BB and TGF-β1. A bidirectional laser trap microrheometry technique was developed that revealed a high degree of local heterogeneity and anisotropy in the structure of the collagen gels during active fibroblast contraction. The use of the growth factors increased not only the gel anisotropy, but the heterogeneity as well, indicating further changes in the collagen fibril orientations. This work shows the ability to influence the remodeling capabilities of fibroblasts by using growth factors in order to begin to elucidate the changes in the local mechanical environment of contracting collagen gels. We present this experimental technique as a method for probing changes in the fibroblast-driven anisotropy of collagen gels as a basis for understanding microstructural tissue organization important in the development of collagen-based tissue equivalents.     

Three-dimensional assessment of lower limb alignment: Reference values and sex-related differences

BackgroundThree-dimensional (3D) preoperative planning and assisted surgery is increasingly popular in deformity surgery and arthroplasty. Reference ranges for 3D lower limb alignment are needed as a prerequisite for standardized analysis of alignment and preoperative planning in 3D, but are not yet established.MethodsOn 60 3D bone models of the lower limbs based on computed tomography data, fifteen parameters per leg were assessed by standardized validated 3D analysis. Distribution parameters and differences between sexes were evaluated. Reference values were generated by adding/subtracting one standard deviation from the mean.ResultsWomen had a significantly lower mean mechanical lateral distal femoral angle compared with men (86.4 ± 2.1° vs. 87.8 ± 2.0°; P < .05) and significantly lower mean joint line convergence angle (−2.5 ± 1.4° vs. -1.3 ± 1.2; P < .01), but higher mean hip knee ankle angle (178.9 ± 1.9° vs. 177.8 ± 2.3°; P < .05) and mean femoral torsion (18.2 ± 9.5° vs. 13.2 ± 6.4°; P < .05), resulting in a tendency towards valgus alignment and vice versa for men. Differences in mean medial proximal tibial angle were not significant. The mean mechanical axis deviation from the tibial knee joint center was 6.9 ± 7.3 mm medial and 1.4 ± 16.1 mm ventral without significant differences between sexes.ConclusionsWe describe total and sex-related reference ranges for all alignment relevant axes and joint angles of the lower limb. There are sex-related differences in certain alignment parameters, which should be considered in analysis and surgical planning.     

Sequential Multimodal Microscopic Imaging and Biaxial Mechanical Testing of Living Multicomponent Tissue Constructs

Understanding relationships between mechanical stimuli and cellular responses require measurements of evolving tissue structure and mechanical properties. We developed a 3D tissue bioreactor that couples to both the stage of a custom multimodal microscopy system and a biaxial mechanical testing platform. Time dependent changes in microstructure and mechanical properties of fibroblast seeded cruciform fibrin gels were investigated while cultured under either anchored (1.0:1.0 stretch ratio) or strip biaxial (1.0:1.1) conditions. A multimodal nonlinear optical microscopy-optical coherence microscopy (NLOM-OCM) system was used to delineate noninvasively the relative spatial distributions of original fibrin, deposited collagen, and fibroblasts during month long culture. Serial in-culture mechanical testing was also performed to track the evolution of bulk mechanical properties under sterile conditions. Over the month long time course, seeded cells and deposited collagen were randomly distributed in equibiaxially anchored constructs, but exhibited preferential alignment parallel to the direction of the 10% stretch in constructs cultured under strip biaxial stretch. Surprisingly, both anchored and strip biaxial stretched constructs exhibited isotropic mechanical properties (including progressively increasing stiffness) despite developing a very different collagen microstructural organization. In summary, our biaxial bioreactor system integrating both NLOM-OCM and mechanical testing provided complementary information on microstructural organization and mechanical properties and, thus, may enable greater fundamental understanding of relationships between engineered soft tissue mechanics and mechanobiology.     

Properties of Collagen in OIM Mouse Tissues

The deletion of the &#102 2 chain from type I collagen in the oim mouse model of osteogenesis imperfecta has been shown to result in a significant reduction in the mechanical strength of the tail tendon and bone tissue. However, the exact role of the &#102 2 chain in reducing the mechanical properties is not clear. We now report that the stabilizing intermolecular cross-links in bone are significantly reduced by 27%, thereby contributing to the loss of tensile strength and the change in stress-strain profile. We also report that, in contrast to previous studies, the denaturation temperature of the triple helical molecule and the intact fibers are 2.6° and 1.9°C higher than the corresponding tail tendon collagen from wild-type mice. The increase in hydroxyproline content accounts, at least in part, for the increase in denaturation temperature. The &#102 2 chain clearly plays an important part in stabilizing the type I collagen triple helix and fiber packing, but further studies are required to determine the precise mechanism.     

Collagen morphology in human meniscal attachments: A SEM study

Qualitative analysis of meniscal attachments from five human knees was completed using scanning electron microscopy (SEM). In addition, quantitative analysis to determine the collagen crimping angle and length in each attachment was done. Morphological differences were revealed between the distinct zones of the attachments from the meniscus transition to the bony insertion. Collagen fibers near to the meniscus appeared inhomogeneous in a radial cross-section view. The sheath surrounding the fibers seemed loose compared with the membrane wrapping around the fibers in the menisci. The midsubstance of human meniscal attachments was composed of collagen fibers running parallel to the longitudinal axis, with a few fibers running obliquely, and others transversely. The bony insertion showed that the crimping pattern vanishes as the collagen fibers approach the fibrocartilagenous enthesis. There were no differences between attachments for crimping angle or length. Collagen crimping angles for all attachments were similar with values of approximately 22°. Crimp length values tended to be smaller for the medial attachments (MA: 4.76 ± 1.95 μm; MP: 3.72 ± 2.31 μm) and higher for the lateral (LA: 6.49 ± 2.34 μm, LP: 6.91 ± 2.29 μm). SEM was demonstrated to be an effective method for revealing the morphology of fibrous connective tissue. The data of collagen fiber length and angle found in this study will allow for better development of microstructural models of meniscal attachments. This study will help to better understand the relation between the morphology and the architecture of collagen and the mechanical behavior of meniscal attachments.