The Essential Role of Fetuin in the Serum-Induced Calcification of Collagen

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. Our goal is to understand the mechanism of fibril mineralization, and as a first step we recently determined the size exclusion characteristics of the fibril. This study indicates that apatite crystals up to 12 unit cells in size can access the water within the fibril while molecules larger than a 40-kDa protein are excluded. We proposed a novel mechanism for fibril mineralization based on these observations, one that relies exclusively on agents excluded from the fibril. One agent generates crystals outside the fibril, some of which diffuse into the fibril and grow, and the other selectively inhibits crystal growth outside of the fibril. We have tested this mechanism by examining the impact of removing the major serum inhibitor of apatite growth, fetuin, on the serum-induced calcification of collagen. The results of this test show that fetuin determines the location of serum-driven mineralization: in fetuin’s presence, mineral forms only within collagen fibrils; in fetuin’s absence, mineral forms only in solution outside the fibrils. The X-ray diffraction spectrum of serum-induced mineral is comparable to the spectrum of bone crystals. These observations show that serum calcification activity consists of an as yet unidentified agent that generates crystal nuclei, some of which diffuse into the fibril, and fetuin, which favors fibril mineralization by selectively inhibiting the growth of crystals outside the fibril.     

Extracellular matrix in disc degeneration

The extracellular matrix of the intervertebral disc structures contains many molecules also found in cartilage. The extremely polyanionic proteoglycans play a central role, particularly in the nucleus, by creating an osmotic environment leading to retention of water and ensuing resistance to deformation-important for the resilience of the tissue. Another major structural entity particularly important in the anulus is the network of collagen fibers; fibril-forming collagen 1 is a major constituent. The collagen fibrils in the anulus are largely oriented in sheets around the nucleus. A number of molecules present in the matrix regulate and direct the collagen fibril assembly by interacting with the collagen molecule and also the formed fibril. Several of these molecules bind by one domain to the collagen fiber and present another functional domain to interact either with other fibers or with other matrix constituents. In this manner the collagen fibers are cross-linked into a network that provides tensile strength and distributes load over large parts of the anulus. Diminished function in these cross-bridging molecules will lead to loss of mechanical properties of the collagen network and result in an impaired ability of the anulus to resist forces delivered by compression of the disc and particularly the nucleus. A different network abundant in the disc and in other load-bearing tissues is based on the beaded filaments of collagen 6. The basic building block is a tetramer of two pairs of antiparallel collagen-6 molecules arranged such that two N-terminal ends of collagen 6 are exposed at either end of the unit. Further assembly occurs both by end-to-end and side-to-side associations. This process is catalyzed by both biglycan and decorin, where the combined effect of direct binding of the core protein to the collagen-6 N-terminal globular domain and the presence of the glycosaminoglycan side chain is essential. These ligands are bound at the same site in complexes extracted from the tissue and then also have one bound molecule of matrilin-1, 2, or 3, in turn bound to a collagen fiber, a procollagen molecule, or an aggrecan. Interactions at the cell surface provide signals to the cells with regard to the conditions of the matrix. Such interactions include binding by matrix components to various receptors at the cell surface. Remodeling of the matrix takes place in response to various factors. An early event in disease is degradation of aggrecan by the members of the ADAMTS (a disintegrin-like and metalloprotease with thrombospondin motifs) family and degradation of molecules important in maintaining the collagen network.     

Comparative analysis of the microstructure of the hamstring tendons: an electron microscopic, histologic, and morphologic study

Semitendinosus and gracilis tendons taken from 25 cadaveric knees were investigated using light and electron microscopy, immunohistochemistry, and morphometry. Thickness of the collagen fibrils, fibril/interstitium ratio, density of blood vessels, density of fibroblasts, and distribution of the collagen fibrils (types I, III, and V collagen and elastic fibers) were analyzed. It was hypothesized that the difference in biomechanical stability between the gracilis and semitendinosus tendons could be reflected by different morphologic features. The results of this study showed that the gracilis tendon, in comparison with the semitendinosus tendon, provides a significantly higher fibril/interstitium ratio and a higher density of collagen III fibers. Conversely, the semitendinosus tendon provides a higher density of blood vessels and collagen I fibers. No differences regarding the density of fibroblasts, thickness of collagen fibrils, and elastic and type V collagen fibers were found. In conclusion, the gracilis tendon graft can provide approximately 15% more collagen than the semitendinosus tendon graft with the same thickness. This fact can play an important role for better biomechanical stability of the gracilis tendon.     

Pyridinium cross-links in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing.

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.     

Lipopolysaccharide: neutralization by polymyxin B shuts down the signaling pathway of nuclear factor kappaB in peripheral blood mononuclear cells, even during activation

BACKGROUND: The anatomy of the anterior abdominal wall plays a most significant role in surgery. Thus the three-dimensional architecture of the collagen fibers in linea alba and rectus sheaths was investigated in 12 human cadavers. MATERIAL AND METHODS: The linea alba was divided into 14 different anatomical segments in the craniocaudal direction. Two-hundred-micrometer-thick, eosin-stained sections from these segments were analyzed by confocal laser scanning microscopy. In this way the direction of the collagen fibers was estimated in the midline of the linea alba and in the medial parts of the rectus sheaths. Width and thickness of the linea alba and thickness of the rectus sheaths were measured. RESULTS: In the ventral rectus sheath essentially oblique fibril bundles intermingle with each other, while the dorsal rectus sheath consists chiefly of transverse fibril bundles. In the linea alba three different zones of fiber orientation follow each other from ventral to dorsal: The lamina fibrae obliquae consists of intermingling oblique fibers. The lamina fibrae transversae contains mainly transverse fibril bundles, while an inconstant, small lamina fibrae irregularium is composed of oblique fibers. Different regions can be distinguished in the craniocaudal course of the linea alba: supraumbilical part, umbilical part, transition zone, and infraarcuate part. CONCLUSIONS: A new model of fiber architecture of the linea alba was developed that describes the fiber architecture as a three-dimensional, highly structured meshwork of collagen fibers. In contrast to former models, no separate lines of decussation of the fibers could be found.     

Collagenous fibril texture of the gliding zone of the human tibialis posterior tendon

Light microscopy and scanning electron microscopy was used to describe the collagenous fibril texture of the wrap around region of the human tibialis posterior tendon in human cadavers. In the region where the tendon wraps around the medial malleolus, the anterior part of the tendon directed towards the pulley had the structure of fibrocartilage. This zone started approximately 25-30 millimeters from the navicular insertion. Scanning electron microscopy of the collagen fibril texture revealed three different layers in the fibrocartilaginous zone. The anterior surface was covered by a meshwork of thin unbanded fibrils with a diameter of app. 30 nm. Beneath the superficial network there was an app. 150 microm thick layer of banded collagen fibrils, These fibrils formed lamella-like bundles which intersected at various angles. The main portion of the collagen fibrils lie below this layer and run in a longitudinal direction. The longitudinal fibrils were divided into bundles by loose connective tissue. The location of the fibrocartilage corresponds to the region where the tibialis posterior tendon wraps around the medial malleolus which serves as a pulley. Clinical significance: Our study documented the existence of a fibrocartilaginous zone with a specific collagen fibril texture that corresponds to a possible site of rupture, as reported by several authors. Due to special collagen fibril texture the fibrocartilage may be more vulnerable to repetitive tensile micro-trauma.     

Spatial organization of types I and II collagen in the canine meniscus.

The meniscus of the knee joint is a fibrocartilage mainly composed of type I collagen and smaller amounts of type II collagen. The distribution of type II collagen in the canine meniscus and its spatial relationship to type I collagen was examined by immunohistochemistry and confocal microscopy. Dorsal and coronal slices of the mid-section of medial and lateral menisci from the knee joints of skeletally mature dogs were predigested with Streptomyces hyaluronate lyase and bacterial Protease enzyme XXIV. Monoclonal antibodies against type I collagen (CP17L) and type II collagen (II-II6B3) and an anti-type II collagen polyclonal antibody (AB759) were employed. The staining for type II collagen in the extracellular matrix of hyaline articular cartilage was diffuse without any identifiable spatial organization. In striking contrast, type II collagen in the fibrocartilage of the meniscus stained as an organized network. Type II collagen was distributed throughout the meniscus with the exception of the outer zone containing the blood vessels. Coronal and dorsal staining of the meniscus showed bundles of circumferential fibrils of type I that colocalized with type II collagen in specific sites. These bundles were enwrapped in a second organizational fibrillar system of types I and II collagen that also colocalized. Bundles of circumferential fibrils appeared in cross-section in coronal sections as dots within the interstitial spaces framed by the network of types I and II collagen of the second system. Confocal overlays showed that types I and II collagens were superimposed, suggesting a close spatial proximity between the two collagens. The cells were confined to the types I and II collagen fibrils that enwrapped the bundles. A striking feature of the radial tie fibers was patches of type II collagen without colocalized type I collagen. Our study reveals a unique network of type II collagen in fibrocartilage of the meniscus that serves as a morphological distinction between fibro- and hyaline cartilage.     

A potential mechanism for age-related declines in patellar tendon biomechanics.

Injuries to soft tissues such as tendons are becoming ever more frequent among the elderly. While increasing levels of activity likely contribute to these injuries, age-related declines in tendon strength may also be important. Whether these declines in biomechanical properties are associated with changes in fibril diameter or collagen type remains in question. In this study, age-related changes were investigated in patellar tendons from young adult rabbits (1-year old, n = 17) and from rabbits at the onset of senescence (4-year old, n = 33). Patellar tendon biomechanics was correlated with both collagen fibril diameter and with the presence of type V collagen, a known regulator of collagen fibril diameter. We hypothesize that (a) aging from I to 4 years results in significant reductions in patellar tendon biomechanical properties, and (b) these age-related declines are associated with smaller fibril diameters and with the presence of type V collagen. Maximum stress declined 25% between I and 4 years of age (100.7 +/- 5.6 MPa and 74.3 +/- 3.4 MPa, respectively, p < 0.0003) (mean +/- SEM) and strain energy density declined 40% (p < 0.001). The distribution of collagen fibrils from 4-year old rabbits was skewed significantly towards smaller diameters compared to fibrils from 1-year old rabbits (p < 0.001). Type V collagen was observed only in the 4-year old rabbit tendons. These correlations suggest that with increasing age after skeletal maturity, type V collagen may help to regulate the assembly and thus diameter of collagen fibrils and thereby adversely affect patellar tendon strength.     

Size and Shape of Mineralites in Young Bovine Bone Measured by Atomic Force Microscopy

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm × 6 nm × 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous channels across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.     

Structural changes in collagen fibrils across a mineralized interface revealed by cryo-TEM

The structure of the mineralized collagen fibril, which is the basic building block of mineralized connective tissues, is critical to its function. We use cryo-TEM to study collagen structure at a well-defined hard–soft tissue interface, across which collagen fibrils are continuous, in order to evaluate changes to collagen upon mineralization. To establish a basis for the analysis of collagen banding, we compared cryo-TEM images of rat-tail tendon collagen to a model based on the X-ray structure. While there is close correspondence of periodicity, differences in band intensity indicate fibril regions with high density but lacking order, providing new insight into collagen fibrillar structure. Across a mineralized interface, we show that mineralization results in an axial contraction of the fibril, concomitant with lateral expansion, and that this contraction occurs only in the more flexible gap region of the fibril. Nevertheless, the major features of the banding pattern are not significantly changed, indicating that the axial arrangement of molecules remains largely intact. These results suggest a mechanism by which collagen fibrils are able to accommodate large amounts of mineral without significant disruption of their molecular packing, leading to synergy of mechanical properties.     

Dynamic changes appearing in collagen fibers during intrinsic tendon repair

Intrinsic healing of severed tendons shows a delay in a gain in breaking strength and the tendon becomes translucent. The cause of tendon translucence was investigated in suture-repaired rat Achilles tendon. The repair site with adjacent translucent tendon were evaluated histologically on day 10 by immunofluorescence and transmission electron microscopy. The healing tendon translucent region by hematoxylin-eosin staining had few inflammatory cells, polarized light birefringence showed thinner collagen fibers, immunofluorescence showed few myofibroblasts, and transmission electron microscopy revealed frayed, irregular thin collagen fibers. During embryogenesis, tendon fibers grow by the addition of discreet collagen fibril segment structures. The speculation is that collagen fibril segment structures are released from collagen fibers within the translucent tendon region for reuse during the regeneration of tendon collagen fibers during intrinsic tendon repair. Healing tendon translucence is related to a decrease in the diameter of collagen fibers by the release of collagen fibril segments within tendon bundles/fascicles.     

The loci of mineral in turkey leg tendon as seen by atomic force microscope and electron microscopy

Transmission electron micrographs of fully mineralized turkey leg tendon in cross-section show the ultrastructure to be more complex than has been previously described. The mineral is divided into two regions. Needlelike-appearing crystallites fill the extrafibrillar volume whereas only platelike crystallites are found within the fibrils. When the speciment is tilted through a large angle, some of the needlelike-appearing crystallites are replaced by platelets, suggesting that the needlelike crystallites are platelets viewed on edge. If so, these platelets have their broad face roughly parallel to the fibril surface and thereby the fibril axis, where the intrafibrillar platelets are steeply inclined to the fibril axis. The projection of the intrafibrillar platelets is perpendicular to the fibril axis. The extrafibrillar volume is at least 60% of the total, the fibrils occupying 40%. More of the mineral appears to be extrafibrillar than within the fibrils. Micrographs of the mineralized tendon in thickness show both needlelike-appearing and platelet crystallites. Stereoscopic views show that the needlelike-appearing crystallites do not have a preferred orientation. From the two-dimensional Fourier transform of a selected area of the cross-sectional image, the platelike crystallites have an average dimension of 58 nm. The needlelike-appearing crystallites have an average thickness of 7 nm. The maximum length is at least 90 nm. Atomic force microscopy (AFM) of unstained, unmineralized turkey leg tendon shows collagen fibrils very much like shadow replicas of collagen in electron micrographs. AFM images of the mineralized tendon show only an occasional fibril. Mineral crystallites are not visible. Because the collagen is within the fibrils, the extrafibrillar mineral must be embedded in noncollagenous organic matter. When the tissue is demineralized, the collagen fibrils are exposed. The structure as revealed by the two modalities is a composite material in which each component is itself a composite. Determination of the properties of the mineralized tendon from the properties of its elements is more difficult than considering the tendon to be just mineral-filled collagen.     

Maturation of composite ligament

A variety of synthetic fibrillar ligaments are being developed and clinically investigated. The tissue response to the variety of polymeric fibrils is similar. The soft-tissue response to carbon fiber braid substituting for lateral collateral ligament in three human knees and for the anterior cruciate ligament in one was examined 12-42 months after surgery. An advanced stage of maturity of the composite structure was particularly evident by dense bundles of collagen in one of the collateral ligaments and in the substitute for the anterior cruciate ligament. Associated with the newly formed collagen the carbon fibril initially formed unique carbon collagen of composite structural units. Eventually, subsidence of the fibroblast response was accompanied by an increase in density and width of the collagen fibers and by a loss of the configuration of the carbon-collagen units. Even the mature collagenous structure was not comparable to the natural ligament.     

Large Deformation Mechanisms,Plasticity, and Failure of an Individual Collagen Fibril With Different Mineral Content

Mineralized collagen fibrils are composed of tropocollagen molecules and mineral crystals derived from hydroxyapatite to form a composite material that combines optimal properties of both constituents and exhibits incredible strength and toughness. Their complex hierarchical structure allows collagen fibrils to sustain large deformation without breaking. In this study, we report a mesoscale model of a single mineralized collagen fibril using a bottom‐up approach. By conserving the three‐dimensional structure and the entanglement of the molecules, we were able to construct finite‐size fibril models that allowed us to explore the deformation mechanisms which govern their mechanical behavior under large deformation. We investigated the tensile behavior of a single collagen fibril with various intrafibrillar mineral content and found that a mineralized collagen fibril can present up to five different deformation mechanisms to dissipate energy. These mechanisms include molecular uncoiling, molecular stretching, mineral/collagen sliding, molecular slippage, and crystal dissociation. By multiplying its sources of energy dissipation and deformation mechanisms, a collagen fibril can reach impressive strength and toughness. Adding mineral into the collagen fibril can increase its strength up to 10 times and its toughness up to 35 times. Combining crosslinks with mineral makes the fibril stiffer but more brittle. We also found that a mineralized fibril reaches its maximum toughness to density and strength to density ratios for a mineral density of around 30%. This result, in good agreement with experimental observations, attests that bone tissue is optimized mechanically to remain lightweight but maintain strength and toughness. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Developmental distribution of collagen type XII in cartilage: association with articular cartilage and the growth plate.

Collagen type XII is a member of the fibril-associated collagens and is characterized by a short triple-helical domain with three extended noncollagenous NC3 domains. Previous studies suggested that collagen XII is a component of cartilage but little is known about its spatial-temporal distribution. This study uses a polyclonal antibody to the purified NC3 domain to investigate its developmental distribution in rat forelimb. Collagen XII was present at the joint interzone on embryonic day 16 (E16d) and restricted to the presumptive articular cartilage by E18d. Labeling of the articular surface intensified as development progressed postnatally (day 1 [1d] to 28d) and extended approximately six cell diameters deep. In juvenile rats, collagen XII antibodies also labeled the longitudinal and transverse septa of stacked chondrocytes in the growth plate. However, collagen XII was not associated at any developmental stage with the cartilaginous secondary ossification center and was only weakly expressed in epiphyseal cartilage. Ultrastructural localization of the NC3 domain epitope showed labeling of the surface of collagen II fibrils both in tissue and in isolated fibrils. The results presented provide further evidence that articular cartilage differs substantially from the underlying epiphyseal cartilage and that different chondrocytic developmental fates are reflected in the composition of their extracellular matrix starting early in development. In addition, collagen XII was distributed in areas of cartilage with more organized fibril orientation and may have a role in promoting alignment or stabilizing such an organization, thereby creating a matrix capable of withstanding load-bearing forces.     

Early anchoring collagen fibers at the bone-tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model.

To clarify the early process of recovery at the bone-tendon interface, we used light microscopy and SEM to examine the process of anchoring of collagen fibers to bone in a canine model. At two weeks, tendon, scar tissue, woven bone and lamellar bone were present at the insertion site. SEM revealed anchoring of collagen fibril bundles of the scar to the woven bone. By 4 weeks, the number of anchoring fibers had increased and a parallel arrangement of fibers was observed. SEM demonstrated deep penetration of fibers into the woven bone layer. In addition, the fibers were observed to project into and intermingle with the scar tissue. By 6 weeks, the anchoring fibers had developed fully and were distributed densely over the interface. SEM also revealed that the collagen fibril bundles in the scar tissue had connected with the collagen fibrils of the woven bone by way of the anchoring bundles. The woven bone was identifiable throughout the early stages of recovery as the interface between soft tissue and hard tissue. Throughout all experimental periods, no staining was observed at the interface of the tendon and bone by Saffranin-O. The formation of woven bone was important during early recovery of the tendon-bone interface prior to the completion of fibrocartilage-mediated insertion.     

Collagen structure regulates fibril mineralization in osteogenesis as revealed by cross-link patterns in calcifying callus.

Although >80% of the mineral in mammalian bone is present in the collagen fibrils, limited information is available about factors that determine a proper deposition of mineral. This study investigates whether a specific collagen matrix is required for fibril mineralization. Calcifying callus from dog tibias was obtained at various times (3-21 weeks) after fracturing. At 3 weeks, hydroxylysine (Hyl) levels were almost twice as high as in control bone, gradually reaching normal levels at 21 weeks. The decrease in Hyl levels can only be the result of the formation of a new collagen network at the expense of the old one. The sum of the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) in callus matched that of bone at all stages of maturation. However, the ratio HP/LP was 2.5-4.5 times higher in callus at 3-7 weeks than in normal bone and was normalized at 21 weeks. Some 40% of the collagen was nonmineralized at the early stages of healing, reaching control bone values (approximately 10%) at 21 weeks. In contrast, only a small increase in callus mineral content from 20.0 to 22.6 (% of dry tissue weight) from week 3 to 21 was seen, indicating that initially a large proportion of the mineral was deposited between, and not within, the fibrils. A strong relationship (r = 0.80) was found between the ratio HP/LP and fibril mineralization; the lower the HP/LP ratio, the more mineralized the fibrils were. Because the HP/LP ratio is believed to be the result of a specific packing of intrafibrillar collagen molecules, this study implies that mineralization of fibrils is facilitated by a specific orientation of collagen molecules in the fibrils.     

Decreased Mechanical Strength and Collagen Content in SPARC‐Null Periodontal Ligament Is Reversed by Inhibition of Transglutaminase Activity

The periodontal ligament (PDL) is a critical tissue that provides a physical link between the mineralized outer layer of the tooth and the alveolar bone. The PDL is composed primarily of nonmineralized fibrillar collagens. Expression of secreted protein acidic and rich in cysteine (SPARC/osteonectin), a collagen‐binding matricellular protein, has been shown to be essential for collagen homeostasis in PDL. In the absence of SPARC, PDL collagen fibers are smaller and less dense than fibers that constitute WT PDL. The aim of this study was to identify cellular mechanisms by which SPARC affected collagen fiber assembly and morphology in PDL. Cross‐linking of fibrillar collagens is one parameter that is known to affect insoluble collagen incorporation and fiber morphology. Herein, the reduction in collagen fiber size and quantity in the absence of SPARC expression was shown to result in a PDL with reduced molar extraction force in comparison to that of WT mice (C57Bl/6J). Furthermore, an increase in transglutaminase activity was found in SPARC‐null PDL by biochemical analyses that was supported by immunohistochemical results. Specifically, collagen I was identified as a substrate for transglutaminase in PDL and transglutaminase activity on collagen I was found to be greater in SPARC‐null tissues in comparison to WT. Strikingly, inhibition of transglutaminase activity in SPARC‐null PDL resulted in increases in both collagen fiber thickness and in collagen content, whereas transglutaminase inhibitors injected into WT mice resulted in increases in collagen fiber thickness only. Furthermore, PDL treated with transglutaminase inhibitors exhibited increases in molar extraction force in WT and in SPARC‐null mice. Thus, SPARC is proposed to act as a critical regulator of transglutaminase activity on collagen I with implications for mechanical strength of tissues. © 2015 American Society for Bone and Mineral Research     

The collagens and glycosaminoglycans of the extracellular matrices secreted by bone marrow stromal cells cultured in vivo in diffusion chambers

When rabbit bone marrow cells are cultured in a diffusion chamber implanted into the peritoneal cavity of an athymic mouse, the stromal cells proliferate, differentiate, and produce tissues that have the morphological features of loose fibrous tissue, woven or chondroid bone, and cartilage. The collagens synthesized during the development of the tissues from 7 to 28 days after implantation were identified using specific antibodies to rabbit types I, II, III, and V and rat type IX collagens, while the glycosaminoglycans were characterized histochemically using the dye, Alcian blue. Fibrous tissue forms in the first week and it contains types I, III, and V collagens and hyaluronan. Bone and cartilage develop within the fibrous tissue from about 12 days onwards. The bone matrix contains types I and V collagens, and chondroitin and keratan sulphates. The cartilaginous matrix contains types II and IX collagens, and chondroitin and keratan sulphates. Small amounts of type III collagen are found in the bone, and types I, III, and V collagens in the cartilage. These are thought to be the remnants of the fibrous matrix and decrease as the matrices mature. It is concluded that the tissue in diffusion chambers, formed by a small number of early precursor cells present in the soft tissues of the endosteum and marrow of young rabbits, contains extracellular matrix macromolecules similar to those found in bone and cartilage.     

Ultrastructural morphometry of anterior cruciate and medial collateral ligaments: an experimental study in rabbits.

This study presents morphometric analyses of collagen subfascicle area fraction and collagen fibril diameter distributions for the anterior cruciate (ACL) and medial collateral (MCL) knee ligaments from transmission electron micrographs of ligament cross sections of five mature, female New Zealand White rabbits. Statistically significant differences in subfascicular area fractions were found between the ACL and MCL (0.89 +/- 0.02, 0.97 +/- 0.01, respectively; p less than 0.001). Mean fibril diameters for the ACL and MCL were also significantly different (0.059 +/- 0.005, 0.085 +/- 0.011 microns, respectively; p less than 0.025). Fibril eccentricity (a measure of parallel alignment of collagen fibrils within the ligaments, defined as the ratio of minor to major axes of elliptical fibril outlines) was 0.89 +/- 0.03 and 0.85 +/- 0.08, respectively, for the ACL and MCL; these data were not significantly different (p greater than 0.1). The relative amount of variation in the pooled fibril diameter data due to variation between animals, ligaments, locations within ligaments, and among fibrils at individual locations are reported. The variation of fibril diameter distributions between the ACL and MCL was substantially greater than the variation between different locations within each ligament cross section as well as between different animals. The structural differences reported may help explain known differences in the biomechanical properties of the ACL and MCL.