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Prostate cancer bone metastasis is still one of the most fatal cancer diagnoses for men. Survival of the circulating prostate tumor cells and their adaptation strategy to survive in the bone niche is the key point to determining metastasis in early cancer stages. The promoter of SFRP2 gene, encoding a WNT signaling modulator, is hypermethylated in many cancer types including prostate cancer. Moreover, SFRP2 can positively regulate osteogenic differentiation in vitro and in vivo. Here, we showed SFRP2 overexpression in the prostate cancer cell line PC3 induces an epithelial mesenchymal transition (EMT), increases the attachment, and modifies the transcriptome towards an osteoblast-like phenotype (osteomimicry) in a collagen 1-dependent manner. Our data reflect a novel molecular mechanism concerning how metastasizing prostate cancer cells might increase their chance to survive within bone tissue. [ABSTRACT FROM AUTHOR]

The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 ( p = 0.0083) and P2 ( p < 0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0 ± 0.66% for suspension cells, 46.4 ± 2.9% for monolayer cells, and 127.64 ± 0.90% for pellets ( p < 0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over in vitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such ex vivo explants might serve as a better model system to mimic clinical situations. [ABSTRACT FROM AUTHOR]

Integrin α10β1 is a collagen-binding integrin expressed on chondrocytes. In order to unravel the role of the α10 integrin during development, we generated mice carrying a constitutive deletion of the α10 integrin gene. The mutant mice had a normal lifespan and were fertile but developed a growth retardation of the long bones. Analysis of the skeleton revealed defects in the growth plate after birth characterized by a disturbed columnar arrangement of chondrocytes, abnormal chondrocyte shape and reduced chondrocyte proliferation. Electron microscopy of growth plates from newborn mice revealed an increased number of apoptotic chondrocytes and reduced density of the collagen fibrillar network compared to these structures in control mice. These results demonstrate that integrin α10β1 plays a specific role in growth plate morphogenesis and function. [ABSTRACT FROM AUTHOR]

Ena/vasodilator-stimulated phosphoprotein (VASP) proteins regulate the geometry of the actin cytoskeleton, thereby influencing cell morphology and motility. Analysis of invertebrate mutants implicates Ena/VASP function in several actin-dependent processes such as axon and dendritic guidance, cell migration, and dorsal closure. In vertebrates, genetic analysis of Ena/VASP function is hindered by the broad and overlapping expression of the three highly related family members Mena (Mammalian enabled), VASP, and EVL (Ena-VASP like). Mice deficient in either Mena or VASP exhibit subtle defects in forebrain commissure formation and platelet aggregation, respectively. In this study, we investigated the consequence of deleting both Mena and VASP. Mena-/- VASP-/- double mutants die perinatally and display defects in neurulation, craniofacial structures, and the formation of several fiber tracts in the CNS and peripheral nervous system. [ABSTRACT FROM AUTHOR]

Β1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of Β1 integrin during skeletogenesis, we inactivated the Β1 integrin gene in chondrocytes. We show here that these mutant mice develop a chondrodysplasia of various severity. Βl-deficient chondrocytes had an abnormal shape and failed to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Statl/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21. Altogether these findings establish that Β1-integrin-dependent motility and proliferation of chondrocytes are mandatory events for endochondral bone formation to occur. [ABSTRACT FROM AUTHOR]

Focuses on a study which reported that mice lacking subsequent elevation of cyclic guanosine monophosphate-dependent kinase I have a very low ability to reproduce. Drugs and chemicals used in the study; In vitro fertilization and embryo culture; Immunohistochemistry; Smooth muscle contraction; Results and discussion.

Studies the role of collagen II in notochord removal and formation of intervertebral discs. Collagen II as a fibril-forming collagen that is mainly expressed in cartilage; Role of collagen II in the formation of normal cartilage in mice, particularly the formation of endochondral bone; Description of notochord formation in mice with defective collagen II.

Informs that mice deficient in type II cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinases (cGKs) were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion. The cGKII-deficient mice also developing dwarfism; Cause of the dwarfism; The role of cGKII in various physiological processes.

Osteoarthritis (OA) is a leading cause of disability and source of societal cost in older adults. These include therapeutic pharmacological and cell-based strategies, as well as biomechanical and biochemical mechanisms in OA pathophysiology, structure to function relationship of the extracellular matrix, molecular and neuronal pathways in OA, biomarkers for OA progression, and metabolism. While the mechanical impact of excess body weight on joints may explain lower limb OA, it is unclear whether type 2 diabetes mellitus (T2DM) is linked to OA outside of excess weight and whether T2DM may play a role in OA pathophysiology. [Extracted from the article]

The integrin receptor family plays important roles in cell-to-cell and cell-to-extracellular matrix interactions through the recruitment of accessory molecules. One of them, the integrin cytoplasmic domain-associated protein-1 (ICAP-1; also known as ITGB1BP1), specifically interacts with the cytoplasmic domain of the β1 integrin subunit and negatively regulates its function in vitro. To address the role of ICAP-1 in vivo, we ablated the Icap-1 gene in mice. We report an unexpected role of ICAP-1 in osteoblast function during bone development. Icap-1-deficient mice suffer from reduced osteoblast proliferation and delayed bone mineralization, resulting in the retarded formation of bone sutures. In vitro studies reveal that primary and immortalized Icap-1-null osteoblasts display enhanced adhesion and spreading on extracellular matrix substrates, probably owing to an increase in β1 integrin activation. Finally, we provide evidence that ICAP-1 promotes differentiation of osteoprogenitors by supporting their condensation through modulating the integrin high affinity state. [ABSTRACT FROM AUTHOR]

Injuries, high altitude, and endurance exercise lead to hypoxic conditions in skeletal muscle and sometimes to hypoxia-induced local tissue damage. Thus, regenerative myoblasts/satellite cells are exposed to different levels and durations of partial oxygen pressure depending on the spatial distance from the blood vessels. To date, it is unclear how hypoxia affects myoblasts proliferation, differentiation, and particularly fusion with normoxic myoblasts. To study this, we investigated how 21% and 2% oxygen affects C2C12 myoblast morphology, proliferation, and myogenic differentiation and evaluated the fusion of normoxic- or hypoxic-preconditioned C2C12 cells in 21% or 2% oxygen in vitro. Out data show that the long-term hypoxic culture condition does not affect the proliferation of C2C12 cells but leads to rounder cells and reduced myotube formation when compared with myoblasts exposed to normoxia. However, when normoxic- and hypoxic-preconditioned myoblasts were differentiated together, the resultant myotubes were significantly larger than the control myotubes. Whole transcriptome sequencing analysis revealed several novel candidate genes that are differentially regulated during the differentiation under normoxia and hypoxia in mixed culture conditions and may thus be involved in the increase in myotube size. Taken together, oxygen-dependent adaption and interaction of myoblasts may represent a novel approach for the development of innovative therapeutic targets. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a mouse in which the Cre recombinase gene has been targeted to exon 1 of the matrilin-1 gene (Matn1) to investigate the origins of articular chondrocytes and the development of the knee joint. Analysis of joints from offspring of Matn1-Cre/R26R crosses demonstrated that articular chondrocytes are derived from cells that have never expressed matrilin-1 whereas the remainder of the chondrocytes in the cartilage anlagen expresses matrilin-1. A band of chondrocytes adjacent to the developing interzone in the E13.5 day knee joint became apparent because these chondrocytes did not turn on expression of matrilin-1 in contrast to the other chondrocytes of the anlagen. The chondrocytes of the presumptive articular surface therefore appear to arise directly from a subpopulation of early chondrocytes that do not activate matrilin-1 expression rather than by redifferentiation from the flattened cells of the interzone. In addition, lineage tracing using both Matn1-Cre/R26R and Col2a1-Cre/R26R lines indicated that non-cartilaginous structures in the knee such as cruciate ligament, synovium and some blood vessels are formed by cells derived from the early chondrocytes of the anlagen. [Copyright &y& Elsevier]

Introduction: Crohn's disease (CD) is characterized by pronounced intestinal fibrosis and severe mucosal damage and conventional animal models are limited to reflect these pathological manifestations. The aim of this study was to examine whether the combination of patient immune-profiling and preclinical studies in a mouse model based on NOD/scid IL-2Rγnull (NSG) reconstituted with peripheral blood mononuclear cells (PBMC) from CD patients has the capacity to harmonize ex vivo human and in vivo animal studies. Methods: Immunological profiles of CD (n = 24) and ulcerative colitis (UC) patients (n = 47) were established by flow cytometry of subgroups of immune cells and subjected to hierarchical cluster and estimation graphics analyses. Pathological phenotypes of NSG mice, which were reconstituted with PBMC from CD, UC, and non-IBD donors (NSG-CD, NSG-UC, and NSG-non-IBD) were compared. Readouts were the clinical, colon, and histological scores; subtypes of immune cells from spleen and colon; and levels of inflammatory markers, such as c-reactive protein (CRP), monocyte chemotactic protein (MCP)-3, transforming growth factor-beta (TGFß), and hepatocyte growth factor (HGF). Fibrocytes were identified by immunohistochemistry in colonic sections. Results: CD patients were significantly clustered in a group characterized by increased levels of TH1, TH2 cells, and decreased levels of CD14+ CD163+ monocytes (p = .003). In contrast to NSG-UC mice, NSG-CD mice exhibited an immune-remodeling phenotype characterized by enhanced collagen deposition, elevated levels of CD14+ CD163+ monocytes, HGF, and TGFß. This phenotype was further corroborated by the presence of human fibrocytes as components of fibrotic areas. Conclusion: The NSG-CD model partially reflects the human disease and allows for studying the development of fibrosis. [ABSTRACT FROM AUTHOR]

Previous anatomical studies have shown different functional zones in human nasal septal cartilage (NC). These zones differ in respect to histological architecture and biochemical composition. The aim of this study was to investigate the influence of these zones on the fate of stem cells from a regenerative perspective. Therefore, decellularized porcine septal cartilage was prepared and subjected to histological assessment to demonstrate its equivalence to human cartilage. Decellularized porcine NC (DPNC) exposed distinct surfaces depending on two different histological zones: the outer surface (OS), which is equivalent to the superficial zone, and the inner surface (IS), which is equivalent to the central zone. Human adipose tissue-derived stem cells (ASCs) were isolated from the abdominal fat tissue of five female patients and were seeded on the IS and OS of DPNC, respectively. Cell seeding efficiency (CSE), vitality, proliferation, migration, the production of sulfated glycosaminoglycans (sGAG) and chondrogenic differentiation capacity were evaluated by histological staining (DAPI, Phalloidin, Live-Dead), biochemical assays (alamarBlue®, PicoGreen®, DMMB) and the quantification of gene expression (qPCR). Results show that cell vitality and CSE were not influenced by DPNC zones. ASCs, however, showed a significantly higher proliferation and elevated expression of early chondrogenic differentiation, as well as fibrocartilage markers, on the OS. On the contrary, there was a significantly higher upregulation of hypertrophy marker MMP13 (p < 0.0001) and GAG production (p = 0.0105) on the IS, whereas cell invasion into the three-dimensional DPNC was higher in comparison to the OS. We conclude that the zonal-dependent distinct architecture and composition of NC modulates activities of ASCs seeded on DPNC. These findings might be used for engineering of cartilage substitutes needed in facial reconstructive surgery that yield an equivalent histological and functional structure, such as native NC. [ABSTRACT FROM AUTHOR]

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues. [ABSTRACT FROM AUTHOR]

Background: Cartilage shortage is a major problem in facial reconstructive surgery. Prior studies have shown that decellularized porcine nasal septal cartilage (DPNC) seeded with primary human nasal chondrocytes enabled cartilage regeneration and showed potential as a replacement material for nasal cartilage. Since adipose tissue‐derived stem cells (ASCs) are easily accessible and almost abundantly available, they appear to be a promising alternative to limited chondrocytes making the combination of DPNC and ASCs a feasible approach towards clinical translation. Thus, this study was intended to investigate the interactions between ASCs and DPNC in an in vitro model. Methods: DPNCs were seeded and 3D‐cultured with primary human ASCs that were priorly characterized with trilineage differentiation and flow cytometry. Cell vitality and proliferation were evaluated by Live‐Dead, alamarBlue, and PicoGreen assays. Chondrogenic differentiation was examined by DMMB assay and cryosectioning‐based histology. Cell invasion within DPNC was visualized and quantified by fluorescent histology (DAPI, Phalloidin). Results: ASCs showed good adherence to DPNC and Live‐Dead assay proved their viability over 2 weeks. AlamarBlueassay showed an increase in metabolic activity compared to 2D cultures, and PicoGreen assay demonstrated an increase of cell number within DPNC over time. Biochemical assays and histology added evidence of chondrogenic differentiation of 3D‐cultured ASCs under the influence of chondrogenic induction medium. Fluorescent image analysis showed a significant increase of cell‐occupied areas of scaffolds over time (P <.05). Conclusions: DPNC scaffolds provided a suitable environment for ASCs that allowed good cell vitality, high proliferation, and chondrogenic differentiation. Thus, the use of ASCs and DPNC yields a promising alternative to the use of primary human chondrocytes. For facial cartilage tissue engineering, we regard ASCs as an attractive alternative to human nasal chondrocytes due to their better accessibility and availability. Further research will be necessary to determine long‐term effects and in vivo outcomes of ASCs and DPNC in cartilage regeneration of the face. [ABSTRACT FROM AUTHOR]

Integrity of the musculoskeletal system is essential for the transfer of muscular contraction force to the associated bones. Tendons and skeletal muscles intertwine, but on a cellular level, the myotendinous junctions (MTJs) display a sharp transition zone with a highly specific molecular adaption. The function of MTJs could go beyond a mere structural role and might include homeostasis of this musculoskeletal tissue compound, thus also being involved in skeletal muscle regeneration. Repair processes recapitulate several developmental mechanisms, and as myotendinous interaction does occur already during development, MTJs could likewise contribute to muscle regeneration. Recent studies identified tendon-related, scleraxis-expressing cells that reside in close proximity to the MTJs and the muscle belly. As the muscle-specific function of these scleraxis positive cells is unknown, we compared the influence of two immortalized mesenchymal stem cell (MSC) lines—differing only by the overexpression of scleraxis—on myoblasts morphology, metabolism, migration, fusion, and alignment. Our results revealed a significant increase in myoblast fusion and metabolic activity when exposed to the secretome derived from scleraxis-overexpressing MSCs. However, we found no significant changes in myoblast migration and myofiber alignment. Further analysis of differentially expressed genes between native MSCs and scleraxis-overexpressing MSCs by RNA sequencing unraveled potential candidate genes, i.e., extracellular matrix (ECM) proteins, transmembrane receptors, or proteases that might enhance myoblast fusion. Our results suggest that musculotendinous interaction is essential for the development and healing of skeletal muscles. [ABSTRACT FROM AUTHOR]

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the β1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wildtype embryos with anti-β1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of β1 integrin for the establishment of PCPand convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations. [ABSTRACT FROM AUTHOR]

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti‐angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament‐specific marker and anti‐angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age‐dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd−/−) mice exhibited more rapid progression of age‐related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd−/−Chm1−/−) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF‐derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age‐related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age‐related IVD degeneration. [ABSTRACT FROM AUTHOR]

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4−/− mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4−/− mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4−/− mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4−/− mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column. [ABSTRACT FROM AUTHOR]

Correct innervation of the main respiratory muscle in mammals, namely the thoracic diaphragm, is a crucial pre-requisite for the functionality of this muscle and the viability of the entire organism. Systemic impairment of Sema3A-Npn-1 (Npn-1 is also known as NRP1) signaling causes excessive branching of phrenic nerves in the diaphragm and into the central tendon region, where the majority of misguided axons innervate ectopic musculature. To elucidate whether these ectopic muscles are a result of misguidance of myoblast precursors due to the loss of Sema3A-Npn-1 signaling, we conditionally ablated Npn-1 in somatic motor neurons, which led to a similar phenotype of phrenic nerve defasciculation and, intriguingly, also formation of innervated ectopic muscles. We therefore hypothesize that ectopic myocyte fusion is caused by additional factors released by misprojecting growth cones. Slit2 and its Robo receptors are expressed by phrenic motor axons and migrating myoblasts, respectively, during innervation of the diaphragm. In vitro analyses revealed a chemoattractant effect of Slit2 on primary diaphragm myoblasts. Thus, we postulate that factors released by motor neuron growth cones have an influence on the migration properties of myoblasts during establishment of the diaphragm. [ABSTRACT FROM AUTHOR]

The growth plate (GP) is a dynamic tissue driving bone elongation through chondrocyte proliferation, hypertrophy and matrix production. The extracellular matrix (ECM) is the major determinant of GP biomechanical properties and assumed to play a pivotal role for chondrocyte geometry and arrangement, thereby guiding proper growth plate morphogenesis and bone elongation. To elucidate the relationship between morphology and biomechanics during cartilage morphogenesis, we have investigated age-dependent structural and elastic properties of the proliferative zone of the murine GP by atomic force microscopy (AFM) from the embryonic stage to adulthood. We observed a progressive cell flattening and arrangement into columns from embryonic day 13.5 until postnatal week 2, correlating with an increasing collagen density and ECM stiffness, followed by a nearly constant cell shape, collagen density and ECM stiffness from week 2 to 4 months. At all ages, we found marked differences in the density and organization of the collagen network between the intracolumnar matrix, and the intercolumnar matrix, associated with a roughly two-fold higher stiffness of the intracolumnar matrix compared to the intercolumnar matrix. This difference in local ECM stiffness may force the cells to arrange in a columnar structure upon cell division and drive bone elongation during embryonic and juvenile development. [ABSTRACT FROM AUTHOR]

Collagen IX (Col IX) is an important component of the cartilage extracellular matrix and has been associated with degenerative cartilage disorders and chondrodysplasias in humans. Further, polymorphisms in Col IX are known risk factors for the development of early intervertebral disc (IVD) degeneration. To understand the role of Col IX in the pathogenesis of IVD disorders, the spine of newborn and older Col IX deficient mice was systematically analyzed and compared to C57BL/6N controls. Morphology and bone parameters of the spine from newborn, 6 and 10 months old animals were investigated using μCT measurements. Histological staining was used to evaluate tissue structure and degree of degeneration. Localization and expression of extracellular matrix proteins was analyzed in depth by immunofluorescence staining, immunoblotting, RT-PCR and in situ hybridization. High resolution imaging and stiffness measurements were performed by atomic force microscopy (AFM). Vertebral bodies of newborn Col IX-deficient mice were smaller and showed an increased mineral density compared to wild type animals. At birth, lack of Col IX led to a disrupted cellular organization in the cartilaginous endplate and a smaller nucleus pulposus of the IVD. Expression levels and localization of other extracellular matrix proteins were strongly altered accompanied by a softening of cartilaginous tissues. In older animals, absence of Col IX caused earlier and more pronounced disc degeneration with annular fissures. The absence of Col IX induces early developmental, structural and biomechanical alterations in both vertebral body and intervertebral disc which eventually cause severe degenerative changes in the aging spine. [ABSTRACT FROM AUTHOR]

In patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE), inflammatory axonal injury is a major determinant of disability; however, the drivers of this injury are incompletely understood. Here, we used the EAE model and determined that the extracellular matrix protein matrilin-2 (MATN2) is an endogenous neuronal molecule that is regulated in association with inflammatory axonal injury. Compared with WT mice, mice harboring a deletion of Matn2 exhibited reduced disease severity and axon damage following induction of EAE. Evaluation of neuron-macrophage cocultures revealed that exogenous MATN2 specifically signals through TLR4 and directly induces expression of proinflammatory genes in macrophages, promoting axonal damage. Moreover, the MATN2-induced proinflammatory response was attenuated greatly in macrophages from Myd88 KO mice. Examination of brain sections from patients with MS revealed that MATN2 is expressed in lesions but not in normal-appearing white matter. Together, our results indicate that MATN2 is a deleterious endogenous neuroaxonal injury response signal that activates innate immune cells and could contribute to early axonal damage in CNS inflammatory diseases like MS. [ABSTRACT FROM AUTHOR]

In patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE), inflammatory axonal injury is a major determinant of disability; however, the drivers of this injury are incompletely understood. Here, we used the EAE model and determined that the extracellular matrix protein matrilin-2 (MATN2) is an endogenous neuronal molecule that is regulated in association with inflammatory axonal injury. Compared with WT mice, mice harboring a deletion of Matn2 exhibited reduced disease severity and axon damage following induction of EAE. Evaluation of neuron-macrophage cocultures revealed that exogenous MATN2 specifically signals through TLR4 and directly induces expression of proinflammatory genes in macrophages, promoting axonal damage. Moreover, the MATN2-induced proinflammatory response was attenuated greatly in macrophages from Myd88 KO mice. Examination of brain sections from patients with MS revealed that MATN2 is expressed in lesions but not in normal-appearing white matter. Together, our results indicate that MATN2 is a deleterious endogenous neuroaxonal injury response signal that activates innate immune cells and could contribute to early axonal damage in CNS inflammatory diseases like MS. [ABSTRACT FROM AUTHOR]

Mast cells, best known as effector cells in pathogenic immunoglobulin-mediated responses, can sense a variety of “danger” signals; if manipulated to enhance their resulting inflammatory responses, they also exacerbate inflammatory diseases such as arthritis and lung inflammation. [ABSTRACT FROM AUTHOR]

Context: Mechanical properties are essential for biological functions of the hyaline cartilage such as energy dissipation and diffusion of solutes. Mechanical properties are primarily dependent on the hierarchical organization of the two major extracellular matrix (ECM) macromolecular components of the cartilage: the fibrillar collagen network and the glycosaminoglycan (GAG)-substituted proteoglycan, mainly aggrecan, aggregates. Interaction of chondrocytes, the only cell type in the tissue, with the ECM through adhesion receptors is involved in establishing mechanical stability via bidirectional transduction of both mechanical forces and chemical signals. In this study, we aimed to determine the role of the transmembrane ß1 integrin adhesion receptors in cartilage biomechanical properties by the use of genetic modification in mice. Methods: Costal cartilages of wild type and mutant mice lacking ß1 integrins in chondrocytes were investigated. Cartilage compressive properties and solute diffusion were characterized by rheometric analysis and Fluorescence Recovery After Photobleaching (FRAP), respectively. Cartilage tissue sections were analyzed by histology, immunohistochemistry and transmission electron microscopy (TEM). Results: At the histological level, the mutant costal cartilage was characterized by chondrocyte rounding and loss of tissue polarity. Immunohistochemistry and safranin orange staining demonstrated apparently normal aggrecan and GAG levels, respectively. Antibody staining for collagen II and TEM showed comparable expression and organization of the collagen fibrils between mutant and control cartilages. Despite the lack of gross histological and ultrastructural abnormalities, rheological measurements revealed that the peak elastic modulus in compression of mutant cartilage was 1.6-fold higher than the peak elastic modulus of wild-type sample. Interestingly, the diffusion coefficient within the mutant cartilage tissue was found to be 1.2-fold lower in the extracellular space and 14-fold lower in the pericellular (PCM) space compared to control. Conclusion: The results demonstrate that the absence of ß1 integrins on the surface of chondrocytes increases the stiffness and modifies the diffusion properties of costal cartilage. Our data imply that ß1 integrins-mediated chondrocyte-matrix interactions directly affect cartilage biomechanics probably by modifying physical properties of individual cells. This study thus highlights the crucial role of ß1 integrins in the cartilage function. [ABSTRACT FROM AUTHOR]

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact- EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation. [ABSTRACT FROM AUTHOR]

Abstract: The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of matrilins and potentially of other extracellular matrix proteins. [Copyright &y& Elsevier]

Abstract: microRNAs (miRNAs) are small non-coding RNAs with fundamental roles in the regulation of gene expression. miRNAs assemble with Argonaute (Ago) proteins to miRNA-protein complexes (miRNPs), which interact with distinct binding sites on mRNAs and regulate gene expression. Specific miRNAs are key regulators of tissue and organ development and it has been shown in mammals that miRNAs are also involved in the pathogenesis of many diseases including cancer. Here, we have characterized the miRNA expression profile of the developing murine genitourinary system. Using a computational approach, we have identified several miRNAs that are specific for the analyzed tissues or the developmental stage. Our comprehensive miRNA expression atlas of the developing genitourinary system forms an invaluable basis for further functional in vivo studies. [ABSTRACT FROM AUTHOR]

The pathological changes in osteoarthritis—a degenerative joint disease prevalent among older people—start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ. [ABSTRACT FROM AUTHOR]

Ageing or obesity are risk factors for protein aggregation in the endoplasmic reticulum (ER) of chondrocytes. This condition is called ER stress and leads to induction of the unfolded protein response (UPR), which, depending on the stress level, restores normal cell function or initiates apoptotic cell death. Here the role of ER stress in knee osteoarthritis (OA) was evaluated. It was first tested in vitro and in vivo whether a knockout (KO) of the protein disulfide isomerase ERp57 in chondrocytes induces sufficient ER stress for such analyses. ER stress in ERp57 KO chondrocytes was confirmed by immunofluorescence, immunohistochemistry, and transmission electron microscopy. Knee joints of wildtype (WT) and cartilage-specific ERp57 KO mice (ERp57 cKO) were analyzed by indentation-type atomic force microscopy (IT-AFM), toluidine blue, and immunofluorescence/-histochemical staining. Apoptotic cell death was investigated by a TUNEL assay. Additionally, OA was induced via forced exercise on a treadmill. ER stress in chondrocytes resulted in a reduced compressive stiffness of knee cartilage. With ER stress, 18-month-old mice developed osteoarthritic cartilage degeneration with osteophyte formation in knee joints. These degenerative changes were preceded by apoptotic death in articular chondrocytes. Young mice were not susceptible to OA, even when subjected to forced exercise. This study demonstrates that ER stress induces the development of age-related knee osteoarthritis owing to a decreased protective function of the UPR in chondrocytes with increasing age, while apoptosis increases. Therefore, inhibition of ER stress appears to be an attractive therapeutic target for OA. [ABSTRACT FROM AUTHOR]

Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. These hydroxylysine-linked carbohydrates were shown recently to be indispensable for the formation of basement membranes (Ruotsalainen, H., Sipilä, L., Vapola, M., Sormunen, R., Salo, A. M., Uitto, L., Mercer, D. K., Robins, S. P., Risteli, M., Aszodi, A., Fässler, R., and Myllylä, R. (2006) J. Cell Sci. 119, 625-635). Analysis of LH3 knock-out embryos and cells in this work indicated that loss of glycosylated hydroxylysines prevents the intracellular tetramerization of type VI collagen and leads to impaired secretion of type IV and VI collagens. Mice lacking the LH activity of LH3 produced slightly underglycosylated type IV and VI collagens with abnormal distribution. The altered distribution and aggregation of type VI collagen led to similar ultrastructural alterations in muscle to those detected in collagen VI knockout and some Ullrich congenital muscular dystrophy patients. Our results provide new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion, assembly, and distribution of highly glycosylated collagen types. [ABSTRACT FROM AUTHOR]

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1/Matn3-/- and Matn3-/- mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins. [ABSTRACT FROM AUTHOR]

Abstract: Type IX collagen is one of the important components, together with type II, V, and XI collagens, in the tectorial membrane of the organ of Corti. To confirm the significance of type IX collagen for normal hearing, we assessed the detailed morphological and electrophysiological features of type IX collagen knock-out mice, which have recently been reported as a deafness model. Through assessment by auditory brainstem response (ABR), knock-out mice were shown to have progressive hearing loss. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape. These morphological changes started in the basal turn and were progressive toward the apical turn. Electron microscopy confirmed disturbance of organization of the collagen fibrils. These results suggest that mutations in type IX collagen genes may lead to abnormal integrity of collagen fibers in the tectorial membrane. [Copyright &y& Elsevier]

The potential of Fourier Transform infrared microspectroscopy (FTIR microspectroscopy) and multivariate analyses were applied for the classification of the frequency ranges responsible for the distribution changes of the main components of articular cartilage (AC) that occur during dietary β-hydroxy-β-methyl butyrate (HMB) supplementation. The FTIR imaging analysis of histological AC sections originating from 35-day old male piglets showed the change in the collagen and proteoglycan contents of the HMB-supplemented group compared to the control. The relative amount of collagen content in the superficial zone increased by more than 23% and in the middle zone by about 17%, while no changes in the deep zone were observed compared to the control group. Considering proteoglycans content, a significant increase was registered in the middle and deep zones, respectively; 62% and 52% compared to the control. AFM nanoindentation measurements collected from animals administered with HMB displayed an increase in AC tissue stiffness by detecting a higher value of Young's modulus in all investigated AC zones. We demonstrated that principal component analysis and artificial neural networks could be trained with spectral information to distinguish AC histological sections and the group under study accurately. This work may support the use and effectiveness of FTIR imaging combined with multivariate analyses as a quantitative alternative to traditional collagenous tissue-related histology. [ABSTRACT FROM AUTHOR]

We have determined the sensitivity and detection limit of a new fiber Bragg grating (FBG)-based optoelectronic micro-indenter for biomechanical testing of cartilage and compared the results to indentation-type atomic force microscopy (IT-AFM) and histological staining. As test samples, we used bovine articular cartilage, which was enzymatically degraded ex vivo for five minutes using different concentrations of collagenase (5, 50, 100 and 500 µg/mL) to mimic moderate extracellular matrix deterioration seen in early-stage osteoarthritis (OA). Picrosirius Red staining and polarization microscopy demonstrated gradual, concentration-dependent disorganization of the collagen fibrillar network in the superficial zone of the explants. Osteoarthritis Research Society International (OARSI) grading of histopathological changes did not discriminate between undigested and enzymatically degraded explants. IT-AFM was the most sensitive method for detecting minute changes in cartilage biomechanics induced by the lowest collagenase concentration, however, it did not distinguish different levels of cartilage degeneration for collagenase concentrations higher than 5 µg/mL. The FBG micro-indenter provided a better and more precise assessment of the level of cartilage degeneration than the OARSI histological grading system but it was less sensitive at detecting mechanical changes than IT-AFM. The FBG-sensor allowed us to observe differences in cartilage biomechanics for collagenase concentrations of 100 and 500 µg/mL. Our results confirm that the FBG sensor is capable of detecting small changes in articular cartilage stiffness, which may be associated with initial cartilage degeneration caused by early OA. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

The application of liposuctioned white adipose tissue (L-WAT) and adipose-derived stem cells (ADSCs) as a novel immunomodulatory treatment option is the currently subject of various clinical trials. Because it is crucial to understand the underlying therapeutic mechanisms, the latest studies focused on the immunomodulatory functions of L-WAT or ADSCs. However, studies that examine the specific transcriptional adaptation of these treatment options to an extrinsic inflammatory stimulus in an unbiased manner are scarce. The aim of this study was to compare the gene expression profile of L-WAT and ADSCs, when subjected to tumor necrosis factor alpha (TNFα), and to identify key factors that might be therapeutically relevant when using L-WAT or ADSCs as an immuno-modulator. Fat tissue was harvested by liposuction from five human donors. ADSCs were isolated from the same donors and shortly subjected to expansion culture. L-WAT and ADSCs were treated with human recombinant TNFα, to trigger a strong inflammatory response. Subsequently, an mRNA deep next-generation sequencing was performed to evaluate the different inflammatory responses of L-WAT and ADSCs. We found significant gene expression changes in both experimental groups after TNFα incubation. However, ADSCs showed a more homogenous gene expression profile by predominantly expressing genes involved in immunomodulatory processes such as CCL19, CCL5, TNFSF15 and IL1b when compared to L-WAT, which reacted rather heterogeneously. As RNA sequencing between L-WAT and ADSCS treated with TNFα revealed that L-WAT responded very heterogeneously to TNFα treatment, we therefore conclude that ADSCs are more reliable and predictable when used therapeutically. Our study furthermore yields insight into potential biological processes regarding immune system response, inflammatory response, and cell activation. Our results can help to better understand the different immunomodulatory effects of L-WAT and ADSCs. [ABSTRACT FROM AUTHOR]

Background: Due to our aging population, an increase in proximal femur fractures can be expected, which is associated with impaired activities of daily living and a high risk of mortality. These patients are also at a high risk to suffer a secondary osteoporosis-related fracture on the contralateral hip. In this context, growth factors could open the field for regenerative approaches, as it is known that, i.e., the growth factor BMP-7 (bone morphogenetic protein 7) is a potent stimulator of osteogenesis. Local prophylactic augmentation of the proximal femur with a BMP-7 loaded thermoresponsive hydrogel during index surgery of an osteoporotic fracture could be suitable to reduce the risk of further osteoporosis-associated secondary fractures. The present study therefore aims to test the hypothesis if a BMP-7 augmented hydrogel is an applicable carrier for the augmentation of non-fractured proximal femurs. Furthermore, it needs to be shown that the minimally invasive injection of a hydrogel into the mouse femur is technically feasible. Methods: In this study, male C57BL/6 mice (n = 36) received a unilateral femoral intramedullary injection of either 100 μl saline, 100 μl 1,4 Butan-Diisocyanat (BDI)-hydrogel, or 100 μl hydrogel loaded with 1 μg of bone morphogenetic protein 7. Mice were sacrificed 4 and 12 weeks later. The femora were submitted to high-resolution X-ray tomography and subsequent histological examination. Results: Analysis of normalized CtBMD (Cortical bone mineral density) as obtained by X-ray micro-computed tomography analysis revealed significant differences depending on the duration of treatment (4 vs 12 weeks; p < 0.05). Furthermore, within different anatomically defined regions of interest, significant associations between normalized TbN (trabecular number) and BV/TV (percent bone volume) were noted. Histology indicated no signs of inflammation and no signs of necrosis and there were no cartilage damages, no new bone formations, or new cartilage tissues, while BMP-7 was readily detectable in all of the samples. Conclusions: In conclusion, the murine femoral intramedullary injection model appears to be feasible and worth to be used in subsequent studies that are directed to examine the therapeutic potential of BMP-7 loaded BDI-hydrogel. Although we were unable to detect any significant osseous effects arising from the mode or duration of treatment in the present trial, the effect of different concentrations and duration of treatment in an osteoporotic model appears of interest for further experiments to reach translation into clinic and open new strategies of growth factor-mediated augmentation. [ABSTRACT FROM AUTHOR]

The gene encoding the proteoglycan aggrecan (Agc1) is abundantly expressed in cartilage during development and adulthood, and the loss or diminished deposition of the protein results in a wide range of skeletal malformations. Furthermore, aggrecan degradation is a hallmark of cartilage degeneration occurring in osteoarthritis. In the present study, we investigated the consequences of a partial loss of aggrecan in the postnatal skeleton and in the articular cartilage of adult mice. We took advantage of the previously described Agc1tm(IRES-CreERT2) mouse line, which allows for conditional and timely-regulated deletion of floxed, cartilage-expressed genes. As previously reported, the introduction of the CreERT2 cassette in the 3'UTR causes a disruption of the normal expression of Agc1 resulting in a hypomorphic deposition of the protein. In homozygous mice, we observed a dwarf phenotype, which persisted throughout adulthood supporting the evidence that reduced aggrecan amount impairs skeletal growth. Homozygous mice exhibited reduced proteoglycan staining of the articular cartilage at 6 and 12 months of age, increased stiffening of the extracellular matrix at six months, and developed severe cartilage erosion by 12 months. The osteoarthritis in the hypomorph mice was not accompanied by increased expression of catabolic enzymes and matrix degradation neoepitopes. These findings suggest that the degeneration found in homozygous mice is likely due to the compromised mechanical properties of the cartilage tissue upon aggrecan reduction. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented related to the use of lifeact peptide to study F-actin dynamics.

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase (LH), hydroxylysyl galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activities in vitro. To investigate the in vivo importance of LH3-catalyzed lysine hydroxylation and hydroxylysine-linked glycosylations, three different LH3-manipulated mouse lines were generated. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that the reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5. Strikingly, survival of hypomorphic embryos and the formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane. Together our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens. [ABSTRACT FROM AUTHOR]

A correction to the article "Early Detection of Aging Cartilage and Osteoarthritis in Mice and Patient Samples Using Atomic Force Microscopy" that was published in the February 1, 2009 issue is presented.

The fibroblast integrin α 11β1 is a key receptor for fibrillar collagens. To study the potential function of α 11 in vivo, we generated a null allele of the α 11 gene. Integrin α 11-/- mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. α 11β1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in α 11-/- cells. We show that α 11β1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that α 11β1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for α 11β1 integrin during tooth eruption. [ABSTRACT FROM AUTHOR]