Correction of Vertebral Bone Development in Ectodysplasin A1-Deficient Mice by Prenatal Treatment With a Replacement Protein.

X-linked hypohidrotic ectodermal dysplasia with the cardinal symptoms hypodontia, hypotrichosis and hypohidrosis is caused by a genetic deficiency of ectodysplasin A1 (EDA1). Prenatal EDA1 replacement can rescue the development of skin appendages and teeth. Tabby mice, a natural animal model of EDA1 deficiency, additionally feature a striking kink of the tail, the cause of which has remained unclear. We studied the origin of this phenomenon and its response to prenatal therapy. Alterations in the distal spine could be noticed soon after birth, and kinks were present in all Tabby mice by the age of 4 months. Although their vertebral bones frequently had a disorganized epiphyseal zone possibly predisposing to fractures, cortical bone density was only reduced in vertebrae of older Tabby mice and even increased in their tibiae. Different availability of osteoclasts in the spine, which may affect bone density, was ruled out by osteoclast staining. The absence of hair follicles, a well-known niche of epidermal stem cells, and much lower bromodeoxyuridine uptake in the tail skin of 9-day-old Tabby mice rather suggest the kink being due to a skin proliferation defect that prevents the skin from growing as fast as the skeleton, so that caudal vertebrae may be squeezed and bent by a lack of skin. Early postnatal treatment with EDA1 leading to delayed hair follicle formation attenuated the kink, but did not prevent it. Tabby mice born after prenatal administration of EDA1, however, showed normal tail skin proliferation, no signs of kinking and, interestingly, a normalized vertebral bone density. Thus, our data prove the causal relationship between EDA1 deficiency and kinky tails and indicate that hair follicles are required for murine tail skin to grow fast enough. Disturbed bone development appears to be partially pre-determined in utero and can be counteracted by timely EDA1 replacement, pointing to a role of EDA1 also in osteogenesis.

Molecular dynamics simulations on human fibulin-4 mutants D203A and E126K reveal conformational changes in EGF domains potentially responsible for enhanced protease lability and impaired extracellular matrix assembly.

Fibulin-4 is a 50 kDa glycoprotein of elastic fibers and plays an important role in development and function of elastic tissues. Fibulin-4 consists of a tandem array of five calcium-binding epidermal growth factor-like modules flanked by N- and C-terminal domains. Mutations in the human fibulin-4 gene EFEMP2 have been identified in patients affected with various arteriopathies including aneurysm, arterial tortuosity, or stenosis, but the molecular basis of most genotype-phenotype correlations is unknown. Here we present biochemical and computer modelling approaches designed to gain further insight into changes in structure and function of two fibulin-4 mutations (E126K and D203A), which are potentially involved in Ca2+ binding in the EGF2 and EGF4 domain, respectively. Using recombinantly produced fibulin-4 mutant and wild type proteins we show that both mutations introduced additional protease cleavage sites, impaired extracellular assembly into fibers, and affected binding to to fibrillin-1, latent TGF-ß-binding proteins, and the lysyl oxidase LOXL2. Molecular dynamics studies indicated that the E126K and D203A mutations do not necessarily result in a direct loss of the complexed Ca2+ ion after 500 ns simulation time, but in significantly enhanced fluctuations within the connecting loop between EGF3 and EGF4 domains and other conformational changes. In contrast, intentionally removing Ca2+ from EGF4 (D203A ΔCa) predicted dramatic changes in the protein structure. These results may explain the changes in protease cleavage sites, reduced secretion and impaired extracellular assembly of the E126K and D203A fibulin-4 mutants and provide further insight into understanding the molecular basis of the associated clinical phenotypes.

A degradation fragment of type X collagen is a real-time marker for bone growth velocity.

Despite its importance as a key parameter of child health and development, growth velocity is difficult to determine in real time because skeletal growth is slow and clinical tools to accurately detect very small increments of growth do not exist. We report discovery of a marker for skeletal growth in infants and children. The intact trimeric noncollagenous 1 (NC1) domain of type X collagen, the marker we designated as CXM for Collagen X Marker, is a degradation by-product of endochondral ossification that is released into the circulation in proportion to overall growth plate activity. This marker corresponds to the rate of linear bone growth at time of measurement. Serum concentrations of CXM plotted against age show a pattern similar to well-established height growth velocity curves and correlate with height growth velocity calculated from incremental height measurements in this study. The CXM marker is stable once collected and can be accurately assayed in serum, plasma, and dried blood spots. CXM testing may be useful for monitoring growth in the pediatric population, especially responses of infants and children with genetic and acquired growth disorders to interventions that target the underlying growth disturbances. The utility of CXM may potentially extend to managing other conditions such as fracture healing, scoliosis, arthritis, or cancer.

SHP2 Regulates the Osteogenic Fate of Growth Plate Hypertrophic Chondrocytes.

Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.

A Dual Role of Upper Zone of Growth Plate and Cartilage Matrix-Associated Protein in Human and Mouse Osteoarthritic Cartilage: Inhibition of Aggrecanases and Promotion of Bone Turnover.

OBJECTIVE: Cartilage damage and subchondral bone changes are closely connected in osteoarthritis. Nevertheless, how these processes are interlinked is, to date, incompletely understood. This study was undertaken to investigate the mechanistic role of a cartilage-derived protein, upper zone of growth plate and cartilage matrix-associated protein (UCMA), in osteoarthritis-related cartilage and bone changes. METHODS: UCMA expression was assessed in healthy and osteoarthritic human and mouse cartilage. For analysis of cartilage and bone changes, osteoarthritis was induced by destabilization of the medial meniscus (DMM) in wild-type (WT) and Ucma-deficient mice. UCMA-collagen interactions, the effect of UCMA on aggrecanase activity, and the impact of recombinant UCMA on osteoclast differentiation were studied in vitro. RESULTS: UCMA was found to be overexpressed in human and mouse osteoarthritic cartilage. DMM-triggered cartilage changes, including increased structural damage, proteoglycan loss, and chondrocyte cell death, were aggravated in Ucma-deficient mice compared to WT littermates, thereby demonstrating the potential chondroprotective effects of UCMA. Moreover, UCMA inhibited ADAMTS-dependent aggrecanase activity and directly interacted with cartilage-specific collagen types. In contrast, osteoarthritis-related bone changes were significantly reduced in Ucma-deficient mice, showing less pronounced osteophyte formation and subchondral bone sclerosis. Mechanistically, UCMA directly promoted osteoclast differentiation in vitro. CONCLUSION: UCMA appears to link cartilage with bone changes in osteoarthritis by supporting cartilage integrity as an endogenous inhibitor of aggrecanases while also promoting osteoclastogenesis and subchondral bone turnover. Thus, UCMA represents an important link between cartilage and bone in osteoarthritis.

ß-catenin activity in late hypertrophic chondrocytes locally orchestrates osteoblastogenesis and osteoclastogenesis.

Trabecular bone formation is the last step in endochondral ossification. This remodeling process of cartilage into bone involves blood vessel invasion and removal of hypertrophic chondrocytes (HTCs) by chondroclasts and osteoclasts. Periosteal- and chondrocyte-derived osteoprogenitors utilize the leftover mineralized HTC matrix as a scaffold for primary spongiosa formation. Here, we show genetically that ß-catenin (encoded by Ctnnb1), a key component of the canonical Wnt pathway, orchestrates this remodeling process at multiple levels. Conditional inactivation or stabilization of ß-catenin in HTCs by a Col10a1-Cre line locally modulated osteoclastogenesis by altering the Rankl:Opg ratio in HTCs. Lack of ß-catenin resulted in a severe decrease of trabecular bone in the embryonic long bones. Gain of ß-catenin activity interfered with removal of late HTCs and bone marrow formation, leading to a continuous mineralized hypertrophic core in the embryo and resulting in an osteopetrotic-like phenotype in adult mice. Furthermore, ß-catenin activity in late HTCs is required for chondrocyte-derived osteoblastogenesis at the chondro-osseous junction. The latter contributes to the severe trabecular bone phenotype in mutants lacking ß-catenin activity in HTCs.

Protein interactions with layers of TiO2 nanotube and nanopore arrays: Morphology and surface charge influence.

In the present work we investigate the key factors involved in the interaction of small-sized charged proteins with TiO2 nanostructures, i.e. albumin (negatively charged), histone (positively charged). We examine anodic nanotubes with specific morphology (simultaneous control over diameter and length, e.g. diameter - 15, 50 or 100nm, length - 250nm up to 10µm) and nanopores. The nanostructures surface area has a direct influence on the amount of bound protein, nonetheless the protein physical properties as electric charge and size (in relation to nanotopography and biomaterial's electric charge) are crucial too. The highest quantity of adsorbed protein is registered for histone, for 100nm diameter nanotubes (10µm length) while higher values are registered for 15nm diameter nanotubes when normalizing protein adsorption to nanostructures' surface unit area (evaluated from dye desorption measurements) - consistent with theoretical considerations. The proteins presence on the nanostructures is evaluated by XPS and ToF-SIMS; additionally, we qualitatively assess their presence along the nanostructures length by ToF-SIMS depth profiles, with decreasing concentration towards the bottom. STATEMENT OF SIGNIFICANCE: Surface nanostructuring of titanium biomedical devices with TiO2 nanotubes was shown to significantly influence the adhesion, proliferation and differentiation of mesenchymal stem cells (and other cells too). A high level of control over the nanoscale topography and over the surface area of such 1D nanostructures enables a direct influence on protein adhesion. Herein, we investigate and show how the nanostructure morphology (nanotube diameter and length) influences the interactions with small-sized charged proteins, using as model proteins bovine serum albumin (negatively charged) and histone (positively charged). We show that the protein charge strongly influences their adhesion to the TiO2 nanostructures. Protein adhesion is quantified by ELISA measurements and determination of the nanostructures' total surface area. We use a quantitative surface charge model to describe charge interactions and obtain an increased magnitude of the surface charge density at the top edges of the nanotubes. In addition, we track the proteins presence on and inside the nanostructures. We believe that these aspects are crucial for applications where the incorporation of active molecules such as proteins, drugs, growth factors, etc., into nanotubes is desired.

Electric Field-Induced Osteogenic Differentiation on TiO2 Nanotubular Layer.

On biocompatible implant surfaces, cellular behavior and fate of stem cells are determined not only by microenvironmental signals but also by electrochemical signals. The potential of electric fields (EFs) to stimulate bone growth and bone healing has been widely demonstrated, but the molecular mechanism linking EFs to osteogenic differentiation has remained elusive. Here we show that constant EFs triggered osteogenic induction of mesenchymal stem cells (MSCs) on a defined nanotubular TiO2 substrate. EFs stimulate the formation of plasma membrane protrusions and the transport of connexin 43 to these protrusions. Connexin 43 is required for the EF-induced lasting intracellular calcium increase, which rapidly propagates to neighboring cells by gap junctions. This enables simultaneous osteogenic induction following downstream calcineurin/CAMKII/NFAT signaling. We propose that connexin 43-mediated, EF-induced osteogenic differentiation of MSCs on a defined nanotubular titanium oxide surface may give new insight on therapeutic interventions for bone regeneration and tissue engineering approaches.

Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa.

Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly. The pathogenetic mechanisms how these mutations translate into the clinical phenotype are, however, poorly understood. In order to elucidate these mechanisms, we expressed fibulin-4 mutants recombinantly in HEK293 cells, purified the proteins in native forms and analyzed alterations in protein synthesis, secretion, matrix assembly, and interaction with other proteins in relation to wild type fibulin-4. Our studies show that different mutations affect these properties in multiple ways, resulting in fibulin-4 deficiency and/or impaired ability to form elastic fibers. The substitutions E126K and C267Y impaired secretion of the protein, but not mRNA synthesis. Furthermore, the E126K mutant showed less resistance to proteases, reduced binding to collagen IV and fibrillin-1, as well as to LTBP1s and LTBP4s. The A397T mutation introduced an extra O-glycosylation site and deleted binding to LTBP1s. We show that fibulin-4 binds stronger than fibulin-3 and -5 to LTBP1s, 3, and 4s, and to the lysyl oxidases LOX and LOXL1; the binding of fibulin-4 to the LOX propeptide was strongly reduced by the mutation E57K. These findings show that different mutations in the fibulin-4 gene result in different molecular defects affecting secretion rates, protein stability, LOX-induced cross-linking, or binding to other ECM components and molecules of the TGF-ß pathway, and thus illustrate the complex role of fibulin-4 in connective tissue assembly.

Loss of fibulin-4 results in abnormal collagen fibril assembly in bone, caused by impaired lysyl oxidase processing and collagen cross-linking.

The extracellular matrix protein fibulin-4 has been shown to be indispensable for elastic fiber assembly, but there is also evidence from human mutations that it is involved in controlling skeletal development and bone stability. Fibulin-4 mutations were identified in patients suffering from vascular abnormality and/or cutis laxa, and some of these patients exhibited bone fragility, arachnodactyly and joint laxity. In order to elucidate the role of fibulin-4 in bone structure and skeletal development, we analyzed structural changes in skeletal tissues of Fbln4(-/-) mice. Immunostaining confirmed that fibulin-4 is highly expressed in cartilage, bone, ligaments and tendons. No morphological abnormalities were found in the skeleton of Fbln4(-/-) mice as compared to wild type littermates except forelimb contractures as well as unusually thick collagen fibrils. Furthermore, fibulin-4 deficiency caused enhanced susceptibility of bone collagen for acid extraction, consistent with significantly reduced lysylpyridinoline and hydroxylysylpyridinoline cross-links in bone. In accordance with that, the amount of lysyl oxidase in long bones and calvaria was strongly decreased and proteolytic activation of lysyl oxidase was reduced in fibulin-4 deficient osteoblasts, while addition of recombinant fibulin-4 rescued the activation. The finding suggested that fibulin-4 is important for the proteolytic activation of lysyl oxidase which has a pivotal role in cross-linking of collagen and elastin.

Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage.

According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond "terminal" differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates - depending on the developmental stage - substantially in trabecular, endosteal, and cortical bone formation. YFP(+) trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP(+)Osx(+) cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate.

Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.

Deletion of beta catenin in hypertrophic growth plate chondrocytes impairs trabecular bone formation.

In order to elucidate the role of ß-catenin in hypertrophic cartilage zone of the growth plate, we deleted the ß-catenin gene ctnnb1specifically from hypertrophic chondrocytes by mating ctnnb1(fl/fl) mice with BAC-Col10a1-Cre-deleter mice. Surprisingly, this resulted in a significant reduction of subchondral trabecular bone formation in BACCol10Cre; ctnnb1(Δ/Δ) (referred to as Cat-ko) mice, although Cre expression was restricted to hypertrophic chondrocytes. The size of the Col10a1 positive hypertrophic zone was normal, but qRT-PCR revealed reduced expression of Mmp13, and Vegfa in Cat-ko hypertrophic chondrocytes, indicating impaired terminal differentiation. Immunohistological and in situ hybridization analysis revealed the substantial deficiency of collagen I positive mature osteoblasts, but equal levels of osterix-positive cells in the subchondral bone marrow space of Cat-ko mice, indicating that the supply of osteoblast precursor cells was not reduced. The fact that in Cat-ko mice subchondral trabeculae were lacking including their calcified cartilage core indicated a strongly enhanced osteoclast activity. In fact, TRAP staining as well as in situ hybridization analysis of Mmp9 expression revealed denser occupation of the cartilage erosion zone with enlarged osteoclasts as compared to the control growth plate, suggesting increased RANKL or reduced osteoprotegerin (Opg) activity in this zone. This notion was confirmed by qRT-PCR analysis of mRNA extracted from cultured hypertrophic chondrocytes or from whole epiphyses, showing increased Rankl mRNA levels in Cat-ko as compared to control chondrocytes, whereas changes in OPG levels were not significant. These results indicate that ß-catenin levels in hypertrophic chondrocytes play a key role in regulating osteoclast activity and trabecular bone formation at the cartilage-bone interface by controlling RANKL expression in hypertrophic chondrocytes.

The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity.

Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment.

Synergistic control of mesenchymal stem cell differentiation by nanoscale surface geometry and immobilized growth factors on TiO2 nanotubes.

The aim of this study is to elucidate whether combined environmental signals provided by nanoscale topography and by growth factors control cell behavior of mesenchymal stem cells (MSCs) in a synergistic or simply additive manner. Chondrogenic and osteogenic differentiation of MSCs is studied on vertically aligned TiO(2) nanotubes of size 15 and 100 nm with and without immobilized bone morphogenetic protein-2 (BMP-2). Although BMP-2 coating stimulates both chondrogenic and osteogenic differentiation of MSCs, the response strongly depends on the surface nanoscale geometry of the BMP-2-coated nanotubes. Chondrogenic differentiation is strongly supported on 100 nm BMP-2-coated nanotubes, but not on 15 nm nanotubes, which induce spreading and de-differentiation of chondrocytes. A similar response is observed with primary chondrocytes, which maintain their chondrogenic phenotype on BMP-2-coated 100 nm nanotubes, but de-differentiate on 15 nm nanotubes. In contrast, osteogenic differentiation is greatly enhanced on 15 nm but not on 100 nm BMP-2-coated nanotubes as shown previously. Furthermore, covalent immobilization of BMP-2 rescues MSCs from apoptosis occurring on uncoated 100 nm TiO(2) nanotube surfaces. Thus, combined signals provided by BMP-2 immobilized to a defined lateral nanoscale spacing geometry seem to contain environmental cues that are able to modulate a lineage-specific decision of MSC differentiation and cell survival in a synergistic manner.

Ucma is not necessary for normal development of the mouse skeleton.

Ucma (Upper zone of growth plate and Cartilage Matrix Associated protein) is a highly conserved tyrosine-sulphated secreted protein of Mw 17 kDa, which is expressed by juvenile chondrocytes. To evaluate the physiological function of this novel cartilage protein, we generated a Ucma-deficient mouse strain by introducing a lacZ/neoR-cassette into the first exon of the Ucma gene. This mutation results in the complete loss of Ucma mRNA and protein expression. Surprisingly, however, although previous in vitro studies implied a role for Ucma in calcification and ossification, these processes were not affected in Ucma-deficient mice during normal development. Likewise, cartilage development was normal. While in previous works Ucma was mainly detected in the cartilage of embryonic and young mice, we detected Ucma expression also in the adult cartilage of the ribs using the lacZ cassette under the control of the Ucma promoter. Moreover, Ucma protein was specifically detected in adult growth plate cartilage by immunohistochemistry. Considering that skeletal development in Ucma-deficient mice is not significantly impaired, protein expression in adult cartilage indicates that Ucma might be involved in skeletal homeostasis and in the mechanical properties of the skeleton during challenging conditions such as ageing or disease.

Covalent functionalization of TiO2 nanotube arrays with EGF and BMP-2 for modified behavior towards mesenchymal stem cells.

In the present work we show the covalent immobilization of two bioactive molecules, epidermal growth factor (EGF) and bone morphogenetic protein-2 (BMP-2), on TiO(2) nanotube surfaces and the resulting influence on the behavior of mesenchymal stem cells. Covalent immobilization of these growth factors onto the oxide surfaces was achieved by N,N-carbonyldiimidazole (CDI) coupling via binding to amine groups of the proteins either directly or via a spacer, namely 11-hydroxy-undecylphosphonic acid (PhoA). The behavior of mesenchymal stem cells can be significantly altered by such an activation procedure. The effect is depending on the diameters of the nanotubes. Most importantly, on 100 nm diameter tubes the cell activity and cell number were drastically increased by grafting such nanotube surfaces with EGF. This demonstrates that the strong diameter dependence on cell activity in the range between 15 and 100 nm observed in prior work can be compensated by coating of the nanotube surfaces with EGF.

Thrombospondin-1 prevents excessive ossification in cartilage repair tissue induced by osteogenic protein-1.

This study investigated the effect of thrombospondin-1 (TSP-1) on the formation of cartilage repair tissue in combination with stimulation by osteogenic protein-1 (OP-1). In miniature pigs, articular cartilage lesions in the femoral trochlea were treated by the microfracture technique and either received no further treatment (MFX), or were treated by additional application of recombinant osteogenic protein-1 (MFX+OP-1), recombinant TSP-1 (MFX+TSP-1), or a combination of both proteins (MFX+TSP-1+OP-1). Six and 26 weeks after surgery, the repair tissue and the degree of endochondral ossification were assessed by histochemical and immunohistochemical methods detecting collagen types I, II, X, TSP-1, and CD31. Microfracture treatment merely induced the formation of inferior fibrocartilaginous repair tissue. OP-1 stimulated chondrogenesis, but also induced chondrocyte hypertrophy, characterized by synthesis of collagen type X, and excessive bone formation. Application of TSP-1 inhibited inadvertant endochondral ossification, but failed to induce chondrogenesis. In contrast, the simultaneous application of both TSP-1 and OP-1 induced and maintained a permanent, nonhypertrophic chondrocyte-like phenotype within cartilage repair tissue. The data of this study demonstrate that OP-1 and TSP-1 complement each other in a functional manner. While OP-1 induces chondrogenesis of the ingrowing cells, TSP-1 prevents their further hypertrophic differentiation and prevents excessive endochondral ossification within the lesions.

Chondromodulin 1 stabilizes the chondrocyte phenotype and inhibits endochondral ossification of porcine cartilage repair tissue.

OBJECTIVE: To investigate the effect of chondromodulin 1 on the phenotype of osteochondral progenitor cells in cartilage repair tissue. METHODS: Self-complementary adeno-associated virus (AAV) vectors carrying chondromodulin 1 complementary DNA (AAV-Chm-1) were applied to cartilage lesions in the knee joints of miniature pigs that were treated by the microfracture technique. Alternatively, isolated porcine osteochondral progenitor cells were infected with AAV-Chm-1 or with AAV-GFP control vectors ex vivo prior to being transplanted into cartilage lesions in which the subchondral bone plate was left intact. The quality of the repair tissue and the degree of endochondral ossification were assessed by histochemical and immunohistochemical methods. The effects of chondromodulin 1 overexpression were also analyzed by angiogenesis assays and quantitative reverse transcriptase-polymerase chain reaction. RESULTS: AAV-Chm-1-infected cells efficiently produced chondromodulin 1, which had strong antiangiogenic effects, as verified by the inhibition of tube formation of endothelial cells. Gene expression analyses in vitro revealed the cell cycle inhibitor p21WAF1/Cip1 as one target up-regulated by AAV-Chm-1. Direct application of AAV-Chm-1 vectors into microfractured porcine cartilage lesions stimulated chondrogenic differentiation of ingrowing progenitor cells, but significantly inhibited terminal chondrocyte hypertrophy, the invasion of vessel structures, and excessive endochondral ossification, which were otherwise observed in untreated lesions. Indirect gene transfer, with infection of porcine osteochondral progenitor cells by AAV-Chm-1 ex vivo, also supported chondrogenic differentiation of these transplanted cells. AAV-Chm-1-infected cells maintained a chondrocyte-like phenotype and formed a hyaline-like matrix that was superior to that formed by uninfected or AAV-GFP-infected cells. CONCLUSION: Our findings indicate that the antiangiogenic factor chondromodulin 1 stabilizes the chondrocyte phenotype by supporting chondrogenesis but inhibiting chondrocyte hypertrophy and endochondral ossification.

Gene expression analysis of ischaemia and reperfusion in human microsurgical free muscle tissue transfer.

The aim of this study was to analyse various gene expression profiles of muscle tissue during normoxia, ischaemia and after reperfusion in human muscle free flaps, to gain an understanding of the occurring regulatory, inflammatory and apoptotic processes on a cellular and molecular basis. Eleven Caucasian patients with soft tissue defects needing coverage with microsurgical free muscle flaps were included in this study. In all patients, the muscle samples were taken from free myocutaneous flaps. The first sample was taken before induction of ischaemia in normoxia (I), another one after ischaemia (II), and the last one was taken after reperfusion (III). The samples were analysed using DNA-microarray, real-time-quantitative-PCR and immunohistochemistry. DNA-microarray analysis detected multiple, differentially regulated genes when comparing the different groups (I-III) with statistical significance. Comparing ischaemia (II) versus normoxia (I) educed 13 genes and comparing reperfusion (III) versus ischaemia (II) educed 19 genes. The comparison of reperfusion (III) versus normoxia (I) yielded 100 differentially regulated genes. Real-time-quantitative-PCR confirmed the results of the DNA-microarrays for a subset of four genes (CASP8, IL8, PLAUR and S100A8). This study shows that ischaemia and reperfusion induces alterations on the gene expression level in human muscle free flaps. Data may suggest that the four genes CASP8, IL8, PLAUR and S100A8 are of great importance in this context. We could not confirm the DNA-microarry and real-time-quantitative-PCR results on the protein level. Finally, these findings correspond with the surgeon's clinical experience that the accepted times of ischaemia, generally up to 90 min., are not sufficient to induce pathophysiological processes, which can ultimately lead to flap loss. When inflammatory and apoptotic proteins are expressed at high levels, flap damage might occur and flap loss is likely. The sole expression on mRNA level might explain why flap loss is unlikely.