Maturation of collagen fibril network structure in tibial and femoral cartilage of rabbits

Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio, Finland.
Osteoarthritis and Cartilage (Impact Factor: 4.17). 11/2009; 18(3):406-15. DOI: 10.1016/j.joca.2009.11.007
Source: PubMed


The structure and composition of articular cartilage change during development and growth, as well as in response to varying loading conditions. These changes modulate the functional properties of cartilage. We studied maturation-related changes in the collagen network organization of cartilage as a function of tissue depth.
Articular cartilage from the tibial medial plateaus and femoral medial condyles of female New Zealand white rabbits was collected from six age-groups: 4 weeks (n=30), 6 weeks (n=30), 3 months (n=24), 6 months (n=24), 9 months (n=27) and 18 months (n=19). Collagen fibril orientation, parallelism (anisotropy) and optical retardation were analyzed with polarized light microscopy. Differences in the development of depth-wise collagen organization in consecutive age-groups and the two joint locations were compared statistically.
The collagen fibril network of articular cartilage undergoes significant changes during maturation. The most prominent changes in collagen architecture, as assessed by orientation, parallelism and retardation were noticed between the ages of 4 and 6 weeks in tibial cartilage and between 6 weeks and 3 months in femoral cartilage, i.e., orientation became more perpendicular-to-surface, and parallelism and retardation increased with changes being most prominent in the deep zone. At the age of 6 weeks, tibial cartilage had a more perpendicular-to-surface orientation in the middle and deep zones than femoral cartilage (P<0.001) and higher parallelism throughout the tissue depth (P<0.001), while femoral cartilage exhibited more parallel-to-surface orientation (P<0.01) above the deep zone after maturation. Optical retardation of collagen was higher in tibial than in femoral cartilage at the ages of 4 and 6 weeks (P<0.001), while at older ages, retardation below the superficial zone in the femoral cartilage became higher than in the tibial cartilage.
During maturation, there is a significant modulation of collagen organization in articular cartilage which occurs earlier in tibial than in femoral cartilage, and is most pronounced in the deep zone.

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    • "The structure of these collagenous tissues can be characterized by small angle X-ray scattering (SAXS) to yield, for example, quantitative measures of fibril orientation and fibril D-spacing [5–7]. While other methods have been used to study collagen fibril orientation including polarized light microscopy [8], reflection anisotropy [9], small angle light scattering [10], confocal laser scattering [11], Raman polarisation [12], and anisotropic Raman scattering [13], synchrotron based SAXS has the advantage of excellent nonsubjective quantification combined with good spatial resolution. "
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    ABSTRACT: Bovine pericardium is used for heart valve leaflet replacement where the strength and thinness are critical properties. Pericardium from neonatal animals (4–7 days old) is advantageously thinner and is considered as an alternative to that from adult animals. Here, the structures of adult and neonatal bovine pericardium tissues fixed with glutaraldehyde are characterized by synchrotron-based small angle X-ray scattering (SAXS) and compared with the mechanical properties of these materials. Significant differences are observed between adult and neonatal tissue. The glutaraldehyde fixed neonatal tissue has a higher modulus of elasticity (83.7 MPa) than adult pericardium (33.5 MPa) and a higher normalised ultimate tensile strength (32.9 MPa) than adult pericardium (19.1 MPa). Measured edge on to the tissue, the collagen in neonatal pericardium is significantly more aligned (orientation index (OI) 0.78) than that in adult pericardium (OI 0.62). There is no difference in the fibril diameter between neonatal and adult pericardium. It is shown that high alignment in the plane of the tissue provides the mechanism for the increased strength of the neonatal material. The superior strength of neonatal compared with adult tissue supports the use of neonatal bovine pericardium in heterografts.1. IntroductionHeart valve leaflet replacement with bovine pericardium is an established practice [1] using either adult or calf pericardium [2] and may be performed percutaneously [3]. It is essential that the mechanical strength and performance of the material are adequate for a long life in service [4]. Greater understanding of the properties of these materials and the structural basis for these properties is important for improving the serviceability of these replacements.Pericardium is a fibrous collagen extracellular matrix material with structural similarities to skin and other tissues. The structure of these collagenous tissues can be characterized by small angle X-ray scattering (SAXS) to yield, for example, quantitative measures of fibril orientation and fibril D-spacing [5–7]. While other methods have been used to study collagen fibril orientation including polarized light microscopy [8], reflection anisotropy [9], small angle light scattering [10], confocal laser scattering [11], Raman polarisation [12], and anisotropic Raman scattering [13], synchrotron based SAXS has the advantage of excellent nonsubjective quantification combined with good spatial resolution.The fact that there is a function-structure relationship between collagen alignment and mechanical strength is well known [14]. The orientation of collagen measured edge on (alignment in plane) has been shown in bovine and ovine skin to be correlated with strength [15, 16]. This correlation extends across a range of mammal species with a strength range of over a factor of five [17]. It is the three-dimensional orientation that is important: simply taking an observation of the fibril orientation normal to the surface of the tissue is not very helpful. Instead, it is necessary to measure the orientation of the fibrils through the thickness of the skin to determine the extent to which they cross between the top and the bottom of the skin layer [17].The orientation of collagen in pericardium heterograft materials for heart valve leaflets has been shown to affect the stiffness during flexing [18]. In ovine and bovine skin, the orientation of the fibrils in the skin influences the mechanical properties [16, 17].We have previously found that pericardium from neonatal calves (4–7 days old) has superior properties for potential application for heart valve repair [19]. Although both adult and calf bovine pericardia are used in heart valve repair, neonatal pericardium has not yet been used for heart valve manufacture. The greater tensile modulus of neonatal pericardium compared to that of adult pericardium may enable the thinner neonatal tissue to be used. This would allow a smaller introducer size for percutaneous heart valves. This makes the application of these heart valves possible through diseased femoral arteries which may have reduced diameters [20]. Glutaraldehyde crosslinked pericardium continues to be the material of choice for heart valve manufacturers and developers. There are several devices on the market and more devices currently in clinical trial that use glutaraldehyde treated tissues.It is known that collagen tissue properties change with age. Differences have been shown in the thermal stability of tendon collagen between steers aged 24–30 months and bulls aged 5 years and this has been attributed to increased level of maturity and thermally stable crosslinks [21]. Glycation of collagen increases with age and can lead to differences in mechanical properties of the collagen. It has been shown to increase stiffness in connective tissues [22] and collagen gels [23] and increase brittleness in bones [24]. Porcine extracellular matrix scaffolds derived from small intestinal submucosa of younger animals and used for in vivo remodeling have been studied previously. They were associated with a more constructive, site appropriate, tissue remodeling response than scaffolds derived from older animals [25]. However, specific physical factors causing this difference were not identified.It has also been found that tissue strength varies with collagen fibril diameter. Larger diameter collagen fibrils are present in stronger tissue. In human aortic valves, the collagen fibril diameter depends on whether the fibrils are from regions of high stress or low stress: larger diameter fibrils (in areas of lower fibril density) result from high stress, suggesting that these larger diameter fibrils provide increased strength [26]. Similarly, for mouse and rat tendon, fibril diameters increase with loading [27, 28]. It is proposed that this is due to the extra mechanical load placed on the tendons on the exercising animals (due to their higher activity levels) stimulating fibril thickening [28]. In bovine leather, fibril diameter is found to be only weakly correlated with strength [29].The size distribution of the fibril diameter has also been found to change with age. Fetal tissue has been found to have a unimodal distribution with smaller collagen fibril diameters, whereas older tissue has larger fibrils and may have a unimodal or bimodal size distribution depending on the tissue type and animal [30]. In studies of equine digital flexor tendons, fibril diameter decreases with exercise, suggesting weakening of tendon with exercise (i.e., fatter fibril is stronger). Unusually, the fibril diameter in these tendons decreases with age, and this is associated with the decrease in strength [31, 32].In the percutaneous delivery of heart valves, the size of the device when folded for delivery is important. Devices made from adult bovine pericardium or porcine pericardium typically require a size 18 F to 25 F catheter (7.0–8.4 mm) [33]. This size is in part dictated by the thickness of the pericardium that is used in the valve, with thicker material folding into a larger diameter device for insertion. A study of 79 patients with peripheral arterial disease found that occluded femoral arteries had an average internal diameter of mm with 12 below 3.5 mm (11 F on the French catheter scale) [20]. These occluded arteries are significantly smaller than the folded heart valves resulting in difficulties for percutaneous delivery of existing heart valve technology. This provides a motivation to find thinner but sufficiently strong material as a substitute for the existing bovine or porcine pericardium. Neonatal pericardium is one possible option that is investigated here.The structural differences between neonatal pericardium and adult tissue that give rise to the desirable differences in their physical properties have not been adequately investigated. This study investigates and compares the collagen fibril structure of neonatal and adult bovine pericardium using SAXS. Specifically, the fibril orientation and the fibril diameter are examined. The use of SAXS at a modern synchrotron facility allows analysis of a small area (250 × 80 μm), enabling quantification of fibril orientation edge on in relatively thin pericardium tissues, a process that is difficult to achieve by other methods.2. Methods Pericardia were selected from 10 adult (18–24 months old) and 10 neonatal (4–7 days old) cattle. The fresh pericardia (less than 72 hours postmortem and typically 48–72 hours postmortem) were washed several times in PBS buffer (pH , 0.01% NaCl). Adult tissue was typically processed closer to 48 hours postmortem while neonatal tissue was processed closer to 72 hours postmortem due to the logistics of obtaining the samples. The tissue was stored at 4–7°C from harvest until the start of washing. Washing in PBS buffer took place at room temperature. The pericardium was then cut and flattened into a “butterfly” shape and held flat with weights around the edge (Figure 1) with care taken to ensure that there were no air bubbles trapped beneath the material.
    Full-text · Article · Sep 2014 · BioMed Research International
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    • "Cartilage also contains chondrocytes, that is, articular cartilage cells, which are surrounded by a thin layer called the pericellular matrix (PCM). The characteristic fibrous structure of the ECM is a result of depth-wise remodeling of the collagen fibril network during maturation [1] [2] [3] [4] and represents a three-layer laminar architecture in adults (Figure 1) [5]. The collagen network is known to be arranged into parallel planes, revealing split-line patterns in cartilage surfaces [5]. "
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    ABSTRACT: The function of articular cartilage depends on its structure and composition, sensitively impaired in disease (e.g. osteoarthritis, OA). Responses of chondrocytes to tissue loading are modulated by the structure. Altered cell responses as an effect of OA may regulate cartilage mechanotransduction and cell biosynthesis. To be able to evaluate cell responses and factors affecting the onset and progression of OA, local tissue and cell stresses and strains in cartilage need to be characterized. This is extremely challenging with the presently available experimental techniques and therefore computational modeling is required. Modern models of articular cartilage are inhomogeneous and anisotropic, and they include many aspects of the real tissue structure and composition. In this paper, we provide an overview of the computational applications that have been developed for modeling the mechanics of articular cartilage at the tissue and cellular level. We concentrate on the use of fibril-reinforced models of cartilage. Furthermore, we introduce practical considerations for modeling applications, including also experimental tests that can be combined with the modeling approach. At the end, we discuss the prospects for patient-specific models when aiming to use finite element modeling analysis and evaluation of articular cartilage function, cellular responses, failure points, OA progression, and rehabilitation.
    Full-text · Article · Apr 2013 · Computational and Mathematical Methods in Medicine
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    • "During growth and maturation , changes within the collagen network architecture take place, altering its orientation and organization, finally forming the arcade-like 'Benninghoff network' (Rieppo et al. 2009; Julkunen et al. 2010). Proteoglycans form the other major group of macromolecules in the ECM. "
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    ABSTRACT: Articular cartilage and subchondral bone act together, forming a unit as a weight-bearing loading-transmitting surface. A close interaction between both structures has been implicated during joint cartilage degeneration, but their coupling during normal growth and development is insufficiently understood. The purpose of the present study was to examine growth-related changes of cartilage mechanical properties and to relate these changes to alterations in cartilage biochemical composition and subchondral bone structure. Tibiae and femora of both hindlimbs from 7- and 13-week-old (each n = 12) female Sprague-Dawley rats were harvested. Samples were processed for structural, biochemical and mechanical analyses. Immunohistochemical staining and protein expression analyses of collagen II, collagen IX, COMP and matrilin-3, histomorphometry of cartilage thickness and COMP staining height were performed. Furthermore, mechanical testing of articular cartilage and micro-CT analysis of subchondral bone was conducted. Growth decreased cartilage thickness, paralleled by a functional condensation of the underlying subchondral bone due to enchondral ossification. Cartilage mechanical properties seem to be rather influenced by growth-related changes in the assembly of major ECM proteins such as collagen II, collagen IX and matrilin-3 than by growth-related alterations in its underlying subchondral bone structure. Importantly, the present study provides a first insight into the growth-related structural, biochemical and mechanical interaction of articular cartilage and subchondral bone. Finally, these data contribute to the general knowledge about the cooperation between the articular cartilage and subchondral bone.
    Full-text · Article · Oct 2012 · Journal of Anatomy
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