Collagen Fibers In Cartilage Research Articles

Experimental study on repair of cartilage defects in the rabbits with GelMA-MSCs scaffold prepared by three-dimensional bioprinting.

Cartilage damage is a common orthopedic disease, which can be caused by sports injury, obesity, joint wear, and aging, and cannot be repaired by itself. Surgical autologous osteochondral grafting is often required in deep osteochondral lesions to avoid the later progression of osteoarthritis. In this study, we fabricated a gelatin methacryloyl-marrow mesenchymal stem cells (GelMA-MSCs) scaffold by three-dimensional (3D) bioprinting. This bioink is capable of fast gel photocuring and spontaneous covalent cross-linking, which can maintain high viability of MSCs and provide a benign microenvironment to promote the interaction, migration, and proliferation of cells. In vivo experiments, further, proved that the 3D bioprinting scaffold can promote the regeneration of cartilage collagen fibers and have a remarkable effect on cartilage repair of rabbit cartilage injury model, which may represent a general and versatile strategy for precise engineering of cartilage regeneration system.

Diffusion tractography of the rat knee at microscopic resolution.

To evaluate whole knee joint tractography, including articular cartilage, ligaments, meniscus, and growth plate using diffusion tensor imaging (DTI) at microscopic resolution. Three rat knee joints were scanned using a modified 3D diffusion-weighted spin echo pulse sequence with 90- and 45-μm isotropic spatial resolution at 9.4T. The b values varied from 250 to 1250 s/mm2 with 4 times undersampling in phase directions. Fractional anisotropy (FA) and mean diffusivity (MD) were compared at different spatial resolution and b values. Tractography was evaluated at multiple b values and angular resolutions in different connective tissues, and compared with conventional histology. The mean tract length and tract volume in various types of tissues were also quantified. DTI metrics (FA and MD) showed consistent quantitative results at 90- and 45-μm isotropic spatial resolutions. Tractography of various connective tissues was found to be sensitive to the spatial resolution, angular resolution, and diffusion weightings. Higher spatial resolution (45 μm) supported tracking the cartilage collagen fiber tracts from the superficial zone to the deep zone, in a continuous and smooth progression in the transitional zone. Fiber length and fiber volume in the growth plate were strongly dependent on angular resolution and b values, whereas tractography in ligaments was found to be less dependent on spatial resolution. High spatial and angular resolution DTI and diffusion tractography can be valuable for knee joint research because of its visualization capacity for collagen fiber orientations and quantitative evaluation of tissue's microscopic properties.

A Shared Epitope of Collagen Type XI and Type II Is Recognized by Pathogenic Antibodies in Mice and Humans with Arthritis.

Collagen XI (CXI) is a heterotrimeric molecule with triple helical structure in which the α3(XI) chain is identical to the α1(II) chain of collagen II (CII), but with extensive posttranslational modifications. CXI molecules are intermingled in the cartilage collagen fibers, which are mainly composed of CII. One of the alpha chains in CXI is shared with CII and contains the immunodominant T cell epitope, but it is unclear whether there are shared B cell epitopes as the antibodies tend to recognize the triple helical structures. Mice expressing the susceptible immune response gene Aq were immunized with CII or CXI. Serum antibody responses were measured, monoclonal antibodies were isolated and analyzed for specificity to CII, CXI, and triple helical collagen peptides using bead-based multiplex immunoassays, enzyme-linked immunosorbent assays, and Western blots. Arthritogenicity of the antibodies was investigated by passive transfer experiments. Immunization with CII or CXI leads to a strong T and B cell response, including a cross-reactive response to both collagen types. Immunization with CII leads to severe arthritis in mice, with a response toward CXI at the chronic stage, whereas CXI immunization induces very mild arthritis only. A series of monoclonal antibodies to CXI were isolated and of these, the L10D9 antibody bound to both CXI and CII equally strong, with a specific binding for the D3 epitope region of α3(XI) or α1(II) chain. The L10D9 antibody binds cartilage in vivo and induced severe arthritis. In contrast, the L5F3 antibody only showed weak binding and L7D8 antibody has no binding to cartilage and did not induce arthritis. The arthritogenic L10D9 antibody bound to an epitope shared with CII, the triple helical D3 epitope. Antibody levels to the shared D3 epitope were elevated in the sera from mice with arthritis as well as in rheumatoid arthritis. CXI is immunologically not exposed in healthy cartilage but contains T and B cell epitopes cross-reactive with CII, which could be activated in both mouse and human arthritis and could evoke an arthritogenic response.

A micro-architecturally biomimetic collagen template for mesenchymal condensation based cartilage regeneration

The unique arcade-like orientation of collagen fibers enables cartilage to bear mechanical loads. In this study continuous-length aligned collagen threads were woven to emulate the interdigitated arcade structure of the cartilage. The weaving pattern provided a macropore network within which micromass cell pellets were seeded to take advantage of mesenchymal condensation driven chondrogenesis. Compression tests showed that the baseline scaffold had a modulus of 0.83±0.39MPa at a porosity of 80%. The modulus of pellet seeded scaffolds increased by 60% to 1.33±0.37MPa after 28days of culture, converging to the modulus of the native cartilage. The scaffolds displayed duress under displacement controlled low-cycle fatigue at 15% strain amplitude such that load reduction stabilized at 8% after 4500 cycles of loading. The woven structure demonstrated a substantial elastic recoil where 40% mechanical strain was close to completely recovered following unloading. A robust chondrogenesis was observed as evidenced by positive staining for GAGs and type II collagen and aggrecan. Dimethyl methylene blue and sircol assays showed GAGs and collagen productions to increase from 3.36±1.24 and 31.46±3.22 at day 3 to 56.61±12.12 and 136.70±12.29μg/μg of DNA at day 28 of culture. This woven collagen scaffold holds a significant potential for cartilage regeneration with shorter in vitro culture periods due to functionally sufficient mechanical robustness at the baseline. In conclusion, the mimicry of cartilage’s arcade architecture resulted in substantial improvement of mechanical function while enabling one of the first pellet delivery platforms enabled by a macroporous network. Statement of significanceMesenchymal condensation is critical for driving chondrogenesis, making high density cell seeding a standard in cartilage tissue engineering. Efforts to date have utilized scaffold free delivery of MSCs in pellet form. This study developed a macroporous scaffold that is fabricated by weaving highly aligned collagen threads. The scaffold can deliver high density cell condensates while providing mechanical stiffness comparable to that of cartilage. The scaffold also mimicked the arcade-like orientation of collagen fibers in cartilage. A highly robust chondrogenesis was observed in this mesenchymal cell pellet delivery system. Baseline mechanical robustness of this scaffold system will enable delivery of cell pellets as early as three days.

Alteration of cartilage surface collagen fibers differs locally after immobilization of knee joints in rats.

The purpose of this study was to examine the ultrastructural changes of surface cartilage collagen fibers, which differ by region and the length of the experimental period in an immobilization model of rat. Male Wistar rats were randomly divided into histological or macroscopic and ultrastructural assessment groups. The left knees of all the animals were surgically immobilized by external fixation for 1, 2, 4, 8 or 16 weeks (n = 5/time point). Sagittal histological sections of the medial mid-condylar region of the knee were obtained and assessed in four specific regions (contact and peripheral regions of the femur and tibia) and two zones (superficial and deep). To semi-quantify the staining intensity of the collagen fibers in the cartilage, picrosirius red staining was used. The cartilage surface changes of all the assessed regions were investigated by scanning electron microscopy (SEM). From histological and SEM observations, the fibrillation and irregular changes of the cartilage surface were more severe in the peripheral region than in the contact region. Interestingly, at 16 weeks post-immobilization, we observed non-fibrous structures at both the contact and peripheral regions. The collagen fiber staining intensity decreased in the contact region compared with the peripheral region. In conclusion, the alteration of surface collagen fiber ultrastructure and collagen staining intensity differed by the specific cartilage regions after immobilization. These results demonstrate that the progressive degeneration of cartilage is region specific, and depends on the length of the immobilization period.

Nano-Morphology of Cartilage in Hydrated and Dehydrated Conditions Revealed by Atomic Force Microscopy

In this study, Atomic Force Microscopy (AFM) was used to examine nano-scale structure difference in superficial and intermediate layers of bovine cartilage under dehydrated and hydrated conditions. The nanostructure of cartilage was quantitatively described using collagen fibril D-periodic spacing, fibril diameter, and surface roughness. Cartilage collagen fiber distribution was inhomogeneous in dehydrated condition but more homogeneous in hydrated condition. AFM images showed that the cartilage in dehydrated condition has higher roughness than that in hydrated condition. This study indicates that water is an important factor in maintaining the structure of extracellular matrix of cartilage. The loss of water can directly result in a rougher structure both on surface and bulk of articular cartilage. The morphology change of hydration-dehydration processes in cartilage is reversible.

Form birefringence as applied to biopolymer and inorganic material supraorganization

I review here form, or textural, birefringence (ΔF) in the context of advances in the field, as well as with regard to findings and applications in the physics of photonic devices, fibers maintaining polarization, photonic crystal fibers, and in biopolymers present in extracellular matrices and the myelin sheath. Some advantages of applying knowledge of ΔF to biological fields involving biopolymers, especially collagen fibers, are considered in more detail. Tendon and cartilage collagen fibers have been regarded as a model of dense, highly aggregated biopolymers with preferential orientations. Owing to their supramolecular organization, such materials may be used to study molecular order by using anisotropic optical properties, especially ΔF. Differences between collagen type I- and collagen type II-rich structures, and similarities between collagen crimp and second harmonic generation images are reported. Based on data reported here, it is possible to deduce that collagen type I supramolecular organization has nonlinear optical properties and that tendon segments can conduct red laser light. With respect to nerve fibers, the detection and measurements of ΔF have allowed the myelin sheath to be considered a smectic liquid crystal.

Towards an analytical model of soft biological tissues

In the past years, soft-tissue modelling research has seen substantial developments, a significant part of which can be ascribed to the refinement of numerical techniques, such as Finite Element analysis. A large class of physico-mechanical properties can be effectively simulated and predictions can be made for a variety of phenomena. However, there is still much that can be conceptually explored by means of fundamental theoretical analysis. In the past few years, driven by our interest in articular cartilage mechanics, we have developed theoretical microstructural models for linear elasticity and permeability that accounted for the presence and arrangement of collagen fibres in cartilage. In this paper, we investigate analytically the non-linear elasticity of soft tissues with collagen fibres arranged according to a given distribution of orientation, a problem that, aside from the case of fibres aligned in a finite number of distinct directions, has been treated exclusively numerically in the literature. We show that, for the case of a tissue with complex fibre arrangement, such as articular cartilage, the theoretical framework commonly used leads to an integral expression of the elastic strain energy potential. The present model is a first attempt in the development of a unified analytical microstructural model for non-linear elasticity and permeability of hydrated, fibre-reinforced soft tissues.

Novel optical imaging technique to determine the 3-D orientation of collagen fibers in cartilage: variable-incidence angle polarization-sensitive optical coherence tomography

To investigate a novel optical method to determine the three dimensional (3-D) structure of articular cartilage collagen non-destructively. Polarization-sensitive optical coherence tomography was used to determine the apparent optical birefringence of articular cartilage for a number of different illumination directions. A quantitative method based on the theory of light propagation in uniaxial crystalline materials was validated on equine flexor tendon. Qualitative maps of fiber polar and azimuthal orientation at sites on the posterior and anterior segments of the equine third metacarpophalangeal (fetlock) joint were produced, and the azimuthal orientations compared with data from a split-line experiment. Polar and azimuthal angles of cut flexor tendon broadly agreed with the nominal values but suggested that the accuracy was limited by our method of determining the apparent birefringence. On intact equine fetlock joints we found a non-zero polar tilt that changed in direction at various points along the apex, moving from the sagittal ridge outwards. The azimuthal orientation changes from being parallel to the sagittal ridge in the posterior region to being inclined to the ridge in the anterior region. This broadly agrees with split-line data for the anterior region but differs in the posterior region, possibly reflecting depth-dependent orientation changes. General quantitative agreement was found between our method and histology in validation experiments. Qualitative results for cartilage suggest a complicated 3-D structure that warrants further study. There is potential to develop this approach into a tool that can provide depth-resolved information on collagen orientation in near real-time, non-destructively and in vivo.

The Factor H Variant Associated with Age-related Macular Degeneration (His-384) and the Non-disease-associated Form Bind Differentially to C-reactive Protein, Fibromodulin, DNA, and Necrotic Cells

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.

Quantitative in situ correlation between microscopic MRI and polarized light microscopy studies of articular cartilage

Objective To establish the correlation between the non-invasive imaging by magnetic resonance microscopy (μMRI) and the histological imaging by polarized light microscopy (PLM) accurately, quantitatively, at the highest possible MRI resolution (13.7μm), and based on the same piece of tissue (articular cartilage from canine shoulder joint).Design In μMRI experiments, the laminar appearance (the magic angle effect) of the proton intensity images and the anisotropic characteristics of the T2relaxation images were analysed. In PLM experiments, the images of the optical retardation and collagen-fibre orientation in cartilage were constructed in two dimensions.Results The T2profile has a distinctly asymmetric bell-shaped curve and three featured zones. The retardation profile has a non-zero minimum at the middle of the transitional zone of the tissue. The angle profile has a smooth variation across the transitional zone. These facts suggest that the collagen fibres in the transitional zone are not entirely random but have a residual order. In addition, the peak of the T2profile coincides with the minimum of the retardation profile, both represent the most isotropic region of the tissue. A hyperbolic tangent function was found to best describe the transition of the collagen fibres in cartilage. A set of criteria was developed for each technique to define the features in the quantitative measurements.Conclusions The criteria offer, for the first time, a set of quantitative and objective means to subdivide the tissue thickness into the zones in histology and in MRI. It is shown that the μMRI zones based on the T2characteristics are statistically equivalent to the histological zones based on the collagen fibre orientation (t-probabilities of 0.730, 0.973, 0.647, 0.850 for the superficial, transitional, radial zones and the total thickness).

The attachment structure of the guinea pig tympanic membrane

Objective: The attachment structure between the tympanic membrane and the surrounding structures, the annular ring and the manubrial part, has not been described fully. We examined the relationship between the tympanic membrane and the tympanic annulus or the handle of the malleus in guinea pigs using light microscopy as well as transmission and scanning electron microscopy (SEM). Methods: We used the temporal bone of the guinea pig and examined the relationship between the tympanic membrane and the surrounding structures using light microscopy and transmission electron microscopy. In the examination of scanning electron microscopy, we used the special method such as alkali–water maceration/SEM method. Results: The results showed that the radial fibers of the tympanic membrane continued directly into the annular ring and the manubrial part. The histological structure of the annular ring was amorphous and collagen fibers were seen in the amorphous matrix. The manubrial part histologically resembled the annular ring. These structures were associated with the cartilage structure. Our observations indicated that the collagen fibers of the tympanic membrane are a special type of cartilage collagen fibers. Conclusion: In conclusion, we propose that the surrounding structures of the tympanic membrane such as the tympanic annulus and manubrial part play an important role in the maintenance and the vibration of the tympanic membrane.

Small-angle HeNe laser light scatter and the compressive modulus of articular cartilage.

Light scattering is a widely used technique for probing the microarchitecture and interactions of biological materials and solutions. In this paper, we describe the use of this method in the study of articular cartilage. The experiments presented utilize small-angle static scattering of HeNe laser light (632.8 nm) from 40 microns thick samples of cartilage taken from the superficial zone of baboon femoral condyle. The specimens were taken from a total of 26 sites in eight animals of various ages. In addition to measuring the dependence of the intensity of scattered light on scatter angle, we performed mechanical testing at the test sites using creep-indentation techniques. The results from the optical and mechanical experiments were compared, and a significant correlation was noted between the average scatter angle and the compressive aggregate modulus. In addition, it was noted that the cartilage of skeletally immature animals had a smaller aggregate modulus and scattered to a higher average angle than the cartilage of skeletally mature animals. A quantitative theory was developed to explain the relation, between mechanical and optical properties in terms of the degree of order in the spatial arrangement of the collagen fibers in cartilage.

Variation of collagen fiber alignment in a joint surface: A scanning electron microscope study of the tibial plateau in dog, rabbit, and man

To determine if articular cartilage collagen fiber organization differs with location on the tibial plateau, specimens from dogs, humans, and rabbits were studied by scanning electron microscopy. Joint surfaces were fixed, dehydrated, and fractured radially so that the periphery could be compared with the center on single specimens. Generally, fibers were more tightly packed in the lateral side than in the medial and the periphery as compared with the center, where the cartilage was consistently thicker and the radial zone was dominant and composed of straight vertical fibers. In the periphery, the tangential and transitional zones were better developed and contributed up to 50% of the cartilage depth in comparison to only 5% centrally. The soft, dull, malacic appearance of the center results from lack of a true surface layer of tangential collagen fibers.

Enthesopathy in the vertebral disc region

In view of the increased numbers of vertebrogenic complaints in society today, the authors investigated the clinical and morphological relations involved in these conditions. Histological techniques and the scanning electron microscope were used to examine the different structures of the spine under conditions of tensile overload. Chief attention was paid to the structures most exposed to mechanical stress, such as the interface between the vertebral body and the intervertebral disc. The authors found that the insertion of collagen fibres in cartilage or bone in the regions overloaded by tensile forces is identical to that found in epicondylitis in other locations. Such enthesopathies in the spinal region may form a major component of back pain states, and special therapy should be employed for them.