Klaus von der Mark

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo

Abstract The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.

TiO2 Nanotube Surfaces: 15 nm—An Optimal Length Scale of Surface Topography for Cell Adhesion and Differentiation†

Studies of biomimetic surfaces in medicine and biomaterial fields have explored extensively how the micrometer-scale topography of a surface controls cell behavior, but only recently has the nanoscale environment received attention as a critical factor for cell behavior. Several investigations of cell interactions have been performed using surface protrusion topographies at the nanoscale; such topographies are typically based on polymer demixing, ordered gold cluster arrays, or islands of adhesive ligands at distinct length scales. Recent work has indicated that the fabrication of ordered TiO2 nanotube layers with controlled diameters can be achieved by anodization of titanium in adequate electrolytes. Such surfaces can almost ideally be used as nanoscale spacing models for size-dependent cellular response. This is particularly important as these studies are carried out on titanium surfaces—a material used for clinical titanium implantations for the purpose of bone, joint, or tooth replacements. Therefore, principles elucidated from this work can guide implant surface modifications toward an optimized surface geometry and profile to best fit and cell interactions for adequate bone growth.

Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix

The ultimate goal in the design of biomimetic materials for use in tissue engineering as permanent or resorbable tissue implants is to generate biocompatible scaffolds with appropriate biomechanical and chemical properties to allow the adhesion, ingrowth, and survival of cells. Recent efforts have therefore focused on the construction and modification of biomimetic surfaces targeted to support tissue-specific cell functions including adhesion, growth, differentiation, motility, and the expression of tissue-specific genes. Four decades of extensive research on the structure and biological influence of the extracellular matrix (ECM) on cell behavior and cell fate have shown that three types of information from the ECM are relevant for the design of biomimetic surfaces: (1) physical properties (elasticity, stiffness, resilience of the cellular environment), (2) specific chemical signals from peptide epitopes contained in a wide variety of extracelluar matrix molecules, and (3) the nanoscale topography of microenvironmental adhesive sites. Initial physical and chemical approaches aimed at improving the adhesiveness of biomaterial surfaces by sandblasting, particle coating, or etching have been supplemented by attempts to increase the bioactivity of biomaterials by coating them with ECM macromolecules, such as fibronectin, elastin, laminin, and collagens, or their integrin-binding epitopes including RGD, YIGSR, and GFOGER. Recently, the development of new nanotechnologies such as photo- or electron-beam nanolithography, polymer demixing, nano-imprinting, compression molding, or the generation of TiO2 nanotubes of defined diameters (15–200 nm), has opened up the possibility of constructing biomimetic surfaces with a defined nanopattern, eliciting tissue-specific cellular responses by stimulating integrin clustering. This development has provided new input into the design of novel biomaterials. The new technologies allowing the construction of a geometrically defined microenvironment for cells at the nanoscale should facilitate the investigation of nanotopography-dependent mechanisms of integrin-mediated cell signaling.

Isolation of a laminin-binding protein from muscle cell membranes.

Skeletal muscle myofibers are each ensheathed by a continuous basal lamina consisting predominantly of type IV collagen, laminin and heparan sulfate proteoglycan. In order to identify laminin‐binding components in the muscle cell surface, plasma membranes from mouse thigh muscle and from rat L6 myoblasts were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred samples with 125I‐labelled laminin revealed a prominent band of approximate mol. wt. 68 000. A protein of this mol. wt. was isolated by affinity chromatography of muscle cell plasma membranes on laminin‐Sepharose. The hydrophobic protein has an apparent mol. wt. of 68 000 and has a high content of serine, glycine and acidic amino acids. After detergent solubilization the purified protein binds to laminin‐coated Sepharose beads at a higher rate than to beads coated with either fibronectin or collagen types I and IV. The interaction of the protein, called LB 68, with laminin was also studied after incorporation into synthetic lecithin vesicles. While detergent‐solubilized LB 68 bound to 125I‐labeled laminin only at lower than physiological ionic strength, liposome‐incorporated LB 68 bound to laminin in the absence of detergents under physiological conditions. We propose that this protein is involved in the interaction of myoblasts with laminin substrates and thus may participate in the anchorage of the basal lamina in the plasmalemma of myotubes.

Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies

For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

Laminin alters cell shape and stimulates motility and proliferation of murine skeletal myoblasts

Proliferating skeletal myoblasts show multiple specific responses to laminin, one of the major glycoprotein components of basement membranes. Using MM14Dy myoblasts, a myogenic cell strain derived from a normal adult mouse skeletal muscle, we show in this study that substrate-bound laminin but not other matrix proteins such as collagens or fibronectin specifically and rapidly induces the outgrowth of cell processes, resulting in bipolar, spindle-shaped cells. This effect is independent from the presence of collagens or serum, and was also observed in primary cultures of fetal mouse skeletal myoblasts. The outgrowth of cell processes on laminin is associated with a dramatic stimulation of cell motility: MM14 myoblasts migrate about five times faster on laminin than on fibronectin. In another series of experiments the effect of laminin and fibronectin on thymidine uptake and proliferation of myoblasts was tested. On top of a type I collagen substrate which was provided to ensure complete adhesion even at low doses of laminin or fibronectin, laminin stimulated myoblast proliferation and incorporation of [3H]thymidine in a dose-dependent manner. The stimulation is two- to threefold higher than on dishes coated with equivalent amounts of fibronectin and is observed both in the presence and in the absence of serum. These results suggest that laminin, a major component of the muscle basal lamina, may be actively involved in the development and regeneration of skeletal muscle.

Synthesis of type iv collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes.

Abstract The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.

Narrow window in nanoscale dependent activation of endothelial cell growth and differentiation on TiO2 nanotube surfaces.

Critical features of biomimetic materials used for vascular grafts and stents are surface structure and chemical features of the implant material supporting adhesion, proliferation, and differentiation of endothelial cells and smooth muscle cells, the major cell types of blood vessels. Recently, experimental evidence from several laboratories have indicated a strong stimulation of cellular activities on vertically aligned TiO(2) nanotube surfaces in comparison to amorphous TiO(2) surfaces. Conflicting reports exist, however, concerning the nanoscale dimension, and the role of the chemistry and crystallinity of the nanotubes in eliciting cell responses. Here we demonstrate that 15 nm nanotubes provide a substantially stronger stimulation of differentiation of mesenchymal cells to endothelial cells and smooth muscle cells than 70-100 nm nanotubes, while high rates of apoptosis were seen on 100 nm nanotubes. Also endothelial cell adhesion, proliferation, and motility were several-fold higher on 15 nm than on 100 nm nanotubes. Furthermore, our data indicate a clear dominance of the nanoscale geometry on endothelial cell behavior over surface chemistry and crystallinity of the TiO(2) nanotube surface. These findings indicate that fine-tuning of TiO(2) surfaces at nanoscale will be an essential parameter in optimizing endothelial cell and smooth muscle cell responses to vascular implants.

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification

SOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

Synovial Stem Cells Are Regionally Specified According to Local Microenvironments After Implantation for Cartilage Regeneration

We previously demonstrated that synovium‐derived MSCs had greater in vitro chondrogenic ability than other mesenchymal tissues, suggesting a superior cell source for cartilage regeneration. Here, we transplanted undifferentiated synovium‐derived MSCs into a full‐thickness articular cartilage defect of adult rabbits and defined the cellular events to elucidate the mechanisms that govern multilineage differentiation of MSCs. Full‐thickness osteochondral defects were created in the knee; the defects were filled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate‐labeled MSCs and covered with periosteum. After 4 weeks, although the cell density decreased, transplanted MSCs produced a great amount of cartilage matrix extensively. The periosteum became thinner, and chondroprogenitors in the periosteum produced a small amount of cartilage matrix. In the deeper zone, transplanted MSCs progressed to the hypertrophic chondrocyte‐like cells. In the deep zone, some transplanted cells differentiated into bone cells and were replaced with host cells thereafter. In the next phase, the border between bone and cartilage moved upwards. In addition, integrations between native cartilage and regenerated tissue were improved. Chondrocyte‐like cells derived from the transplanted MSCs still remained at least after 24 weeks. Histological scores of the MSC group improved continuously and were always better than those of two other control groups. Immunohistological analyses and transmission electron microscopy confirmed that the MSCs produced abundant cartilage matrix. We demonstrated that transplanted synovium‐derived MSCs were altered over a time course according to the microenvironments. Our results will advance MSC‐based therapeutic strategies for cartilage injury and provide the clues for the mechanisms that govern multilineage differentiation of MSCs.