Elastic tissue disruption is a major pathogenic factor to human vascular disease.

Elastic fibers are essential components of the arterial extracellular matrix. They consist of the protein elastin and an array of microfibrils that support the protein and connect it to the surrounding matrix. The elastin gene encodes tropoelastin, a protein that requires extensive cross-linking to become elastin. Tropoelastin is expressed throughout human life, but its expression levels decrease with age, suggesting that the potential to synthesize elastin persists during lifetime although declines with aging. The initial abnormality documented in human atherosclerosis is fragmentation and loss of the elastic network in the medial layer of the arterial wall, suggesting an imbalance between elastic fiber injury and restoration. Damaged elastic structures are not adequately repaired by synthesis of new elastic elements. Progressive collagen accumulation follows medial elastic fiber disruption and fibrous plaques are formed, but advanced atherosclerosis lesions do not develop in the absence of prior elastic injury. Aging is associated with arterial extracellular matrix anomalies that evoke those present in early atherosclerosis. The reduction of elastic fibers with subsequent collagen accumulation leads to arterial stiffening and intima-media thickening, which are independent predictors of incident hypertension in prospective community-based studies. Arterial stiffening precedes the development of hypertension. The fundamental role of the vascular elastic network to arterial structure and function is emphasized by congenital disorders caused by mutations that disrupt normal elastic fiber production. Molecular changes in the genes coding tropoelastin, lysyl oxidase (tropoelastin cross-linking), and elastin-associated microfibrils, including fibrillin-1, fibulin-4, and fibulin-5 produce severe vascular injury due to absence of functional elastin.

Nonlinear microscopy of common histological stains reveals third harmonic generation harmonophores.

Since its invention over a hundred years ago, histological analysis using coloured dye staining remains the gold standard for histopathology. While these stains provide critical information for a variety of diagnostic purposes, they offer limited two-dimensional histological information. Extending classical histological analysis to three dimensions requires novel imaging approaches such as multiphoton microscopy. Multiphoton microscopy enables multimodal, three-dimensional imaging of histologically stained samples. Specifically, third harmonic generation (THG), a nonlinear optical process in which three incident photons are combined into one by the sample, allows high contrast imaging of tissues stained with absorbing dyes, which in turn act as harmonophores. While this technique has previously been applied to hematoxylin and eosin (H&E) tissue sections, we extend this approach to other commonly used histological stains to demonstrate further potential applications of the technique. We demonstrate THG imaging of both human skin and liver tissue stained with H&E, Verhoeff-Van Gieson (VVG) and Picrosirius Red stains. We find that these stains provide excellent contrast as THG harmonophores, enabling high resolution imaging of histological samples. THG imaging of the Verhoeff stain enables easy detection of elastic fibers while Picrosirius Red acts as an effective harmonophore for imaging collagen fibers of all sizes.

Distribution of Elastic Fibers in the Lacrimal Sac and Nasolacrimal Duct of Japanese Cadavers.

PURPOSE: To investigate the distribution of elastic fibers in lacrimal sac and nasolacrimal duct of Japanese cadavers. METHODS: We examined 8 lacrimal sacs and nasolacrimal ducts of 7 Japanese cadavers (4 right sides, 4 left sides; 3 males, 4 females; average age of 89 years at death) that were preserved with 10% buffered formalin. The harvested specimens were transversely sectioned. All specimens were stained with Elastica van Gieson. Micrographs were taken, and then converted to white and black images, and the elastic fiber density of 4 locations (middle and upper levels of lacrimal sac, and middle and lower levels of nasolacrimal duct) was compared. RESULTS: Elastic fibers showed greater distribution in the lamina propria, compared with the submucosal tissue, at all locations (p < 0.001). Elastic fiber density within the lamina propria at the middle lacrimal sac level (13.5% ± 4.3%) was similar to the density observed at the upper lacrimal sac level (13.2% ± 2.3%, p = 0.87) and the lower nasolacrimal duct level (17.8% ± 6.0%, p = 0.088), but was significantly greater than the density observed at the middle nasolacrimal duct level (9.5% ± 3.8%, p = 0.0001). CONCLUSIONS: Elastic fibers in the lacrimal sac and nasolacrimal duct are primarily distributed in the lamina propria, with a greater density at the upper and middle lacrimal sac levels, and at the lower nasolacrimal duct level, compared with the middle nasolacrimal duct level. This distribution of elastic fibers likely reflects the magnitude of mechanical stress within the lacrimal drainage system.

Elastic Fibers and Large Artery Mechanics in Animal Models of Development and Disease.

Development of a closed circulatory system requires that large arteries adapt to the mechanical demands of high, pulsatile pressure. Elastin and collagen uniquely address these design criteria in the low and high stress regimes, resulting in a nonlinear mechanical response. Elastin is the core component of elastic fibers, which provide the artery wall with energy storage and recoil. The integrity of the elastic fiber network is affected by component insufficiency or disorganization, leading to an array of vascular pathologies and compromised mechanical behavior. In this review, we discuss how elastic fibers are formed and how they adapt in development and disease. We discuss elastic fiber contributions to arterial mechanical behavior and remodeling. We primarily present data from mouse models with elastic fiber deficiencies, but suggest that alternate small animal models may have unique experimental advantages and the potential to provide new insights. Advanced ultrastructural and biomechanical data are constantly being used to update computational models of arterial mechanics. We discuss the progression from early phenomenological models to microstructurally motivated strain energy functions for both collagen and elastic fiber networks. Although many current models individually account for arterial adaptation, complex geometries, and fluid-solid interactions (FSIs), future models will need to include an even greater number of factors and interactions in the complex system. Among these factors, we identify the need to revisit the role of time dependence and axial growth and remodeling in large artery mechanics, especially in cardiovascular diseases that affect the mechanical integrity of the elastic fibers.

Histological and Electron Microscope Staining for the Identification of Elastic Fiber Networks.

Elastic fibers are a major component of the extracellular matrix and are present in many tissues. Routine histology and standard electron microscopy procedures often do not allow for clear identification of elastic fibers making their organization and ultrastructure difficult to study. In this paper, we describe staining methods and procedures to enhance the contrast of elastin at both the light and electron microscope levels.

Anogenital Mammary-Like Glands: A Study of Their Normal Histology With Emphasis on Glandular Depth, Presence of Columnar Epithelial Cells, and Distribution of Elastic Fibers.

The normal histology of anogenital mammary-like glands (AGMLG) has been studied previously, but some aspects, including glandular depth, presence of columnar epithelium resembling columnar cell change/hyperplasia as defined in mammary pathology, and distribution of elastic fibers, have not been previously investigated. To address these issues, we studied 148 AGMLG identified in 133 paraffin blocks sampled from 64 vulvar wide excision or vulvectomy specimens (64 patients, various indications for surgery). The depth of AGMLG ranged from 0.64 to 3.9 mm. Epithelial columnar cell change was noted in 33.1% of all AGMLG, whereas columnar cell hyperplasia was detected in 10.1%. Occasionally, combinations of cuboidal epithelium and columnar cell change were seen within 1 histological section. Of 22 specimens stained for elastic fibers, in only 6 (27.3%) cases were elastic fibers found around glands. Periductal elastic fibers were demonstrated around 3 of the only 5 ducts, which were available for analysis in slides stained for elastic fibers. The depth of AGMLG should be taken into account when planning topical and surgical therapies for lesions derived or evolving from AGMLG. Alterations identical to columnar cell change may represent a normal variation of AGMLG.

Biaxial Stretch Improves Elastic Fiber Maturation, Collagen Arrangement, and Mechanical Properties in Engineered Arteries.

Tissue-engineered blood vessels (TEVs) are typically produced using the pulsatile, uniaxial circumferential stretch to mechanically condition and strengthen the arterial grafts. Despite improvements in the mechanical integrity of TEVs after uniaxial conditioning, these tissues fail to achieve critical properties of native arteries such as matrix content, collagen fiber orientation, and mechanical strength. As a result, uniaxially loaded TEVs can result in mechanical failure, thrombus, or stenosis on implantation. In planar tissue equivalents such as artificial skin, biaxial loading has been shown to improve matrix production and mechanical properties. To date however, multiaxial loading has not been examined as a means to improve mechanical and biochemical properties of TEVs during culture. Therefore, we developed a novel bioreactor that utilizes both circumferential and axial stretch that more closely simulates loading conditions in native arteries, and we examined the suture strength, matrix production, fiber orientation, and cell proliferation. After 3 months of biaxial loading, TEVs developed a formation of mature elastic fibers that consisted of elastin cores and microfibril sheaths. Furthermore, the distinctive features of collagen undulation and crimp in the biaxial TEVs were absent in both uniaxial and static TEVs. Relative to the uniaxially loaded TEVs, tissues that underwent biaxial loading remodeled and realigned collagen fibers toward a more physiologic, native-like organization. The biaxial TEVs also showed increased mechanical strength (suture retention load of 303 ± 14.53 g, with a wall thickness of 0.76 ± 0.028 mm) and increased compliance. The increase in compliance was due to combinatorial effects of mature elastic fibers, undulated collagen fibers, and collagen matrix orientation. In conclusion, biaxial stretching is a potential means to regenerate TEVs with improved matrix production, collagen organization, and mechanical properties.

Elastin-based biomaterials and mesenchymal stem cells.

Elastin is the dominant mammalian elastic protein found in soft tissue. Elastin-based biomaterials have the potential to repair elastic tissues by improving local elasticity and providing appropriate cellular interactions and signaling. Studies that combine these biomaterials with mesenchymal stem cells have demonstrated their capacity to also regenerate non-elastic tissue. Mesenchymal stem cell differentiation can be controlled by their immediate environment, and their sensitivity to elasticity makes them an ideal candidate for combining with elastin-based biomaterials. With the growing accessibility of the elastin precursor, tropoelastin, and elastin-derived materials, the amount of research interest in combining these two fields has increased and, subsequently, is leading to the realization of a potentially new strategy for regenerative medicine.

Impact of cyclic stretch on induced elastogenesis within collagenous conduits.

In vitro tissue engineering of vascular conduits requires a synergy between several external factors, including biochemical supplementation and mechanotranductive stimulation. The goal of this study was to improve adult human vascular smooth muscle cell orientation and elastic matrix synthesis within 3D tubular collagen gel constructs. We used a combination of elastogenic factors (EFs) previously tested in our lab, along with cyclic circumferential strains at low amplitude (2.5%) delivered at a range of frequencies (0.5, 1.5, and 3 Hz). After 21 days of culture, the constructs were analyzed for elastic matrix outcomes, activity of matrix metalloproteinases (MMPs)-2 and -9, cell densities and phenotype, and mechanical properties of constructs. While cell densities remained unaffected by the addition of stretch, contractile phenotypic markers were elevated in all stretched constructs relative to control. Constructs cultured with EFs stretched at 1.5 Hz exhibited the maximum elastin mRNA expression and total matrix elastin (over sixfold vs. the static EFs control). MMP-2 content was comparable in all treatment conditions, but MMP-9 levels were elevated at the higher frequencies (1.5 and 3 Hz). Minimal circumferential orientation was achieved and the mechanical properties remained comparable among the treatment conditions. Overall, constructs treated with EFs and stretched at 1.5 Hz exhibited the most elastogenic outcomes.

A new objective histological scale for studying human photoaged skin.

BACKGROUND: A quantitative understanding of the histological alteration of the skin is important for assessing the severity of photoaging. METHODS: We performed Elastica-van Gieson staining and immunohistochemistry for decorin on 34 facial skin sections. We evaluated the alteration of collagen fibers and decorin (a modulator for collagen fibrillogenesis), according to the 5 grades of morphological change in elastic fibers that was established by Kligman (1969). The objectivity of a stage (Stages I-VI), which was established in this study, was evaluated using weighted kappa statistical analysis based on the degree of agreement in stage determination by 11 observers using a blind procedure. Correlation between the crow's-feet-area wrinkles grades of another 26 women and stages was also analyzed. RESULTS: The initial alteration of elastic fibers was observed in the deep dermis. Decorin was not detected in very severely altered skin. Based on the combination of changes in the elastic fibers, collagenic fibers, and decorin, skin tissues were categorized into 6 stages according to severity. The statistical analysis showed almost perfect agreement between observers. Significant positive correlation between stages and wrinkle scores was found. CONCLUSIONS: We propose a new objective histological scale that is useful for assessing the severity of photoaging.

Stereological study of the elastic fiber and smooth muscle cell system in the bovine and buffalo penis / Estereologia do sistema elástico e das células musculares lisas no pênis fibroelástico zebuino e bubalino

Samples of ten penises of Mediterranean buffaloes and ten penises of Red Sindhi cattle were used. The thickness of the tunica albuginea (TA), distribution of smooth muscle cells (SMC) and volume density (Vv) of elastic system fibers in TA, corpus cavernosum (CC) and corpus spongiosum (CS) were evaluated. The Vv of elastic system fibers in buffalo and bovine penis was respectively 4.07% ±0.88% and 3.36% ±1.21% in TA; 17.32% ±2.21% and 13.14% ±1.27% (CC), 26.58% ±4.31% and 31.36% ±3.67% (CS). The CC of buffalo presented higher Vv of elastic fibers than bovine, while in the CS the Vv of elastic fibers in buffaloes was smaller than in cattle. The TA thickness showed a significant difference among the species studied. The arrangement of SMC in the bovine penises and in the water buffalo suggests that this pattern is common to animals that have fibroelastic penises.

O pênis dos zebuinos e bubalinos são classificados como do tipo fibroelástico, sendo grande parte de sua rigidez conferida pelas fibras colágenas e elásticas presentes na matriz extracelular. Embora alguns estudos tenham abordado estes componentes em bovinos europeus, os dados na literatura sobre zebuínos e bubalinos são escassos. Nós avaliamos a espessura da túnica albugínea, a distribuição das células musculares lisas e a densidade volumétrica (Vv) das fibras do sistema elásticona túnica albugínea, no corpo cavernoso e esponjoso do pênis de animais jovens adultos inteiros.Foram utilizados 10 fragmentos de pênis de búfalos da raça Mediterrâneo e 10 fragmentos de pênis de bovinos da raça Red Sindi. Os fragmentos de pênis foram processados de acordo com a técnica histológica de rotina para inclusão em parafina e corados pela Fucsina Resorcina de Weigert, Picro Sirius Red, Tricrômio de Gomori e anticorpo anti α actina. As fibras elásticas foram determinadas em 25 campos aleatórios de cada fragmento, utilizando o sistema teste modelo M42. A Vv das fibras do sistema elástico no pênis do búfalo e bovino foram(média e desvio padrão respectivamente) de 4,07% ±0,88 e 3,36% ±1,21 na túnica albugínea; 17,32% ±2,21 e 13,14% ±1,27 no corpo cavernoso; 26,58% ±4,31 e 31,36% ±3,67 no corpo esponjoso, respectivamente. Houve diferença na Vv das fibras elásticas entre as duas espécies, no corpo cavernoso e no corpo esponjoso. Na espessura da túnica albugínea houve diferença significativa entre o búfalo Mediterrâneo (1,4mm ±0,11) e o bovino Red Sindi (1,76mm ±0,23). Nas duas espécies, as fibras musculares lisas foram encontradas apenas associadas ao endotélio de revestimento dos seios cavernosos. Com base nestes resultados, podemos concluir que existem diferenças na proporção entre os componentes da matriz extracelular nas espécies estudadas, fato que deve ser considerado nos procedimentos clínicos e cirúrgicos veterinários e continua a ser investigado.

Segmentation features and structural organization of the intrapulmonary artery of the yak.

This study aims to systematically investigate intrapulmonary artery segmentation, blood vessel wall characteristics and structure organization, and the interrelation between intrapulmonary artery structure and plateau hypoxia adaptation in yak. The normal intrapulmonary artery structure of the yak had been studied using histological methods and transmission electron microscopy. The intrapulmonary artery of the yak was also examined using morphometric analysis and angiography. Results showed that the elastic intrapulmonary artery is divided into two types, namely, classical and transitional elastic segments. The muscular intrapulmonary artery is divided into three types, namely, transitional, classical muscular, and muscular arteriole segments. In the transitional elastic artery, elastic fibers and smooth muscles are linked through three models of ends, lateral branches, and branch tops. Two phenomena are possible for the transition from the elastic intrapulmonary artery to the muscular artery. One phenomenon postulates that a less elastic membrane is first increased and then suddenly decreased, and another supposes that the elastic membrane is gradually reduced and assembled in one to two layers before entering the transitional muscular artery. The smooth muscle of the intrapulmonary artery tunica media had more apophysis; it was physically connected with elastic membrane or fiber and composed of functionally resilient unit of the intrapulmonary arterial wall. Glycogenosomes increased in the muscular intrapulmonary artery smooth muscle cells. It exist one to two layers intact smooth muscle in intrapulmonary arteriole, the presence of intact smooth muscle in the intrapulmonary arteriole of the yak is a kind of structure adaptation to low-oxygen environment.

Elastin fibers display a versatile microfibril network in articular cartilage depending on the mechanical microenvironments.

Elastin fibers are major extracellular matrix macromolecules that are critical in maintaining the elasticity and resilience of tissues such as blood vessels, lungs and skins. However, the role of elastin in articular cartilage is poorly defined. The present study investigated the organization of elastin fiber in articular cartilage, its relationship to collagen fibers and the architecture of elastin fibers from different mechanical environments by using a kangaroo model. Five morphologies of elastin fibers were identified: Straight fiber, straight fiber with branches, branching fibers directly associated with chondrocyte, wave fiber and fine elastin. The architecture of the elastin network varied significantly with cartilage depth. In the most superficial layer of tibial plateau articular cartilage, dense elastin fibers formed a distinctive cobweb-like meshwork which was parallel to the cartilage surface. In the superficial zone, elastin fibers were well organized in a preferred orientation which was parallel to collagen fibers. In the deep zone, no detectable elastin fiber was found. Moreover, differences in the organization of elastin fibers were also observed between articular cartilage from the tibial plateau, femoral condyle, and distal humerus. This study unravels the detailed microarchitecture of elastin fibers which display a well-organized three-dimensional versatile network in articular cartilage. Our findings imply that elastin fibers may play a crucial role in maintaining the integrity, elasticity, and the mechanical properties of articular cartilage, and that the local mechanical environment affects the architectural development of elastin fibers.

Elastic fiber assembly in the adult mouse pubic symphysis during pregnancy and postpartum.

Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.

Elastic fibers in the aortic valve spongiosa: a fresh perspective on its structure and role in overall tissue function.

This study characterizes the elastic fiber structure within the aortic valve spongiosa, the middle layer of the tri-laminate leaflet. The layer is rich in glycosaminoglycans and proteoglycans, through which it resists compression and lubricates shear between the outer layers. Elastin in this layer forms a fine, interweaving structure, yet it is unclear how this particular structure, which uses elasticity to preload the leaflet, assists spongiosa function. In this study, immunohistochemistry (IHC) and scanning electron microscopy (SEM) are used to characterize spongiosa elastin, as well as investigate regional differences in structure. IHC for elastin highlights an intermediate structure which varies in thickness and density between regions. In particular, the spongiosa elastin is thicker in the hinge and coaptation region than in the belly. SEM of NaOH-digested leaflets shows a rectilinear pattern of elastic fibers in the hinge and coaptation region, as opposed to a radially oriented stripe pattern in the belly. In conclusion, elastic fibers in the spongiosa connect the two outer layers and vary regionally in structure, while possibly playing a role in responding to regionally specific loading patterns.

Whitnall ligament anatomy revisited.

PURPOSE: To examine the concentrations of elastic fibres in Whitnall ligament and the intermuscular transverse ligament (ITL), and to examine the anatomic relationships between Whitnall ligament and its surrounding structures. DESIGN: Observational anatomic study. SAMPLES: Full thickness sagittal sections of 12 upper eyelids (6 right and 6 left), fixed in 10 % buffered formalin, were studied in 10 Asian cadavers (mean age at death: 77.1 years, range: 62-92 years). METHODS: We analysed the concentrations of elastic fibres in Whitnall ligament and the ITL, as well as examined the relationship between Whitnall ligament and the connective tissue septa, the ITL and the levator palpebrae superioris (LPS) muscle. MAIN OUTCOME MEASURES: Concentrations of elastic fibres in Whitnall ligament and the ITL, relationship between Whitnall ligament and its surrounding structures. RESULTS: The concentration of elastic fibres in Whitnall ligament was low compared to the ITL. In most specimens, both Whitnall ligament and the ITL were loosely attached to the LPS muscle through areas of adipose tissue or cavities. Although in all specimens, connective tissue septa were demonstrated next to Whitnall ligament, they were more often directed parallel to the ligament rather than radially to it. CONCLUSION: Both Whitnall ligament, with its low concentration of elastic fibres, and the ITL, with its high content of elastic fibres, show loose attachments to the LPS muscle through areas of adipose tissue or cavities. Whitnall ligament may work as the check ligament of the LPS muscle as well as part of the pulley system with the ITL.

Age-related changes in the hyoepiglottic ligament: functional implications based on histopathologic study.

PURPOSE: The study aimed to identify age-related changes in the hyoepiglottic ligament associated with function of the epiglottis during swallowing and respiration. MATERIALS AND METHODS: Normal postmortem laryngeal tissue samples were obtained at autopsy from 20 individuals with no history of laryngeal disease. The subjects were divided into 2 groups: those aged 81-91 years (elderly group, n = 11) and those aged 31-48 years (non-elderly group, n = 9). Specimens were subjected to Elastica van Gieson and hematoxylin-eosin staining, and characteristics of the hyoepiglottic ligament were compared between groups. RESULTS: The hyoepiglottic ligament extended from the epiglottis to both lingual muscles and the hyoid bone (pars lingualis and pars hyoideus). The numbers of muscle fibers (P < .001), collagenous fibers (P < .01), and elastic fibers (P < .001) were significantly decreased in the elderly group in comparison to those in the non-elderly group. CONCLUSION: Age-related changes in the hyoepiglottic ligament appear to be associated with aspiration, obstructive sleep apnea syndrome, and acquired laryngomalacia in the elderly.

Heterogeneous long-range correlated deformation of semiflexible random fiber networks.

The deformation of dense random fiber networks is important in a variety of applications including biological and nonliving systems. In this paper it is shown that semiflexible fiber networks exhibit long-range power-law spatial correlations of the density and elastic properties. Hence, the stress and strain fields measured over finite patches of the network are characterized by similar spatial correlations. The scaling is observed over a range of scales bounded by a lower limit proportional to the segment length and an upper limit on the order of the fiber length. If the fiber bending stiffness is reduced below a threshold, correlations are lost. The issue of solving boundary value problems defined on large domains of random fiber networks is also addressed. Since the direct simulation of such systems is impractical, the network is mapped into an equivalent continuum with long-range correlated elastic moduli. A technique based on the stochastic finite element method is used to solve the resulting stochastic continuum problem. The method provides the moments of the distribution function of the solution (e.g., of the displacement field). It performs a large dimensionality reduction which is based on the scaling properties of the underlying elasticity of the material. Two examples are discussed in closure.

Stretching stimulates fibulin-5 expression and controls microfibril bundles in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: The elastic fiber system comprises oxytalan, elaunin and elastic fibers, differing in their relative microfibril and elastin contents. Human periodontal ligaments contain oxytalan fibers (pure microfibrils). Periodontal ligaments are continuously exposed to various functional forces, such as tooth movement and occlusal loading. We have reported that bundles of microfibrils coalesce in response to mechanical strain in cultured periodontal ligament fibroblasts, as assessed in terms of their positivity for fibrillin-1 (the major component of microfibrils). However, the mechanism of microfibril coalescence is unclear. We hypothesized that the fibrillin-1-binding molecule, fibulin-5, contributes to oxytalan fiber formation under mechanical strain. MATERIAL AND METHODS: We subjected periodontal ligament fibroblasts to stretching in order to examine the effects of fibulin-5 on the formation of oxytalan fibers in cell/matrix layers. We transfected periodontal ligament cells with small interference RNA for fibulin-5, then examined oxytalan fibers using immunofluorescence and electron microscopy. RESULTS: Immunofluorescence showed that fibrillin-1-positive microfibrils coalesced as a result of stretching, compared with cells that were not subjected to stretching. Fibulin-5 colocalized on fibrillin-1-positive microfibrils. Stretching increased fibulin-5 gene expression and protein deposition. Immunofluorescence and immunogold electron microscopy analysis revealed that fibulin-5 suppression inhibited the coalescence of microfibrils under stretching conditions. CONCLUSION: These results suggest that fibulin-5 up-regulated in response to tension strain may control the formation of microfibril bundles in periodontal ligament.