Daniel J. Hartmann

Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium hydroxide-containing cement

The cement produced microcrystals of calcite by reaction with culture medium supplemented with calf serum. Human dental pulp cells seeded on such a substrate preferentially adhered and aggregated around the microcrystals. Immunofluorescence and immunogold labelling revealed a high affinity of serum fibronectin molecules for the calcite crystals. At 4 weeks in culture, the cells had various features of differentiated odontoblasts, notably nuclear polarization, typical appearance of the Golgi apparatus, synthesis of type I collagen and absence of type III, and apical accumulation of actin and vimentin. These cells also elaborated a collagenous extracellular matrix which did not mineralize.

Biochemical properties and immunolocalization of minor collagens in foetal calf cartilage

Type II collagen is the major collagen of cartilage. However, new collagenous chains have been described in different cartilaginous tissues, recently. The la, 2a and 3a chains were extracted from human and bovine hyaline cartilage [1]. Other minor collagenous components were extracted from neonatal pig and human cartilage noted M, CFI, CF2 [2-4], from bovine nasal cartilage and human intervertebral disc noted CPS1, CPS2 [5,6] and from chicken sternal cartilage noted HMW, LMW [7,8] and M 1, M 2 [9]. Here, we report the partial characterization of 3 collagenous fractions obtained after limited pepsin treatment of foetal calf cartilage and isolated according to their solubility properties. The 1.2 M NaC1 fraction contains the la, 2t~ and 3a chains. The 2.0 M NaC1 and 3.0 M NaCl fractions contain at least 7 collagenous chains, which are related to the disulfide-bonded new chains enumerated above, but show some major differences, Antibodies against the 1.2 M and 2.0 M NaC1 fractions were raised in rabbits and their specificity tested by radioimmunoprecipitation. The localization of the corresponding collagenous chains was achieved by indirect immunofluorescence in epiphyseal proper and growth cartilage from foetal calf cartilage. 2. MATERIALS AND METHODS

Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen

The different collagen types were extracted sequentially, by 4 M guanidinium chloride and pepsin, from human foetal and normal and osteoarthritic adult articular cartilage. They were characterized by electrophoresis and immunoblotting. Most of the collagenous proteins present in articular cartilage from young human foetuses were solubilized: almost 40% of the total collagen was extracted in the native form with 4 M guanidinium chloride. Type VI collagen was detected in this fraction as high-molecular-mass chains (185-220 kDa) and a low-molecular-mass chain (140 kDa). Type II, IX and XI collagens were also present, but were extracted more extensively by pepsin digestion. Comparative analysis of normal and osteoarthritic cartilage from adults reveals some major differences: an increase in the solubility of the collagen and modifications of soluble collagen types in osteoarthritic cartilage. Furthermore, type VI collagen was present at a higher concentration in guanidinium chloride extracts of osteoarthritic cartilage than those of normal tissue. This finding was corroborated by electron microscopic observations of the same samples: abundant (100 nm) periodic fibrils were observed in the disorganized pericellular capsule of cloned cells in osteoarthritic cartilage. In normal tissues the pericellular zone was more compact and contained only a few such banded fibrils. The differences in the collagen types solubilized from normal and osteoarthritic cartilage, although corresponding to a minor proportion of the total collagen, demonstrate that important modifications in chondrocyte metabolism and in the collagenous network do occur in degenerated cartilage.

Immunofluorescent localization of collagen types I and III, and of fibronectin during feather morphogenesis in the chick embryo.

Abstract Collagen types I and III were purified from the skin of 3- or 7-week chickens and fibronectin from human serum. Antibodies were raised in rabbits and used in indirect immunofluorescence on frozen sections of 5- to 16-day chick embryo feather-forming skin. Prior to the formation of dense feather-forming dermis, anticollagen fluorescence was confined to a thin underlining of the dermal-epidermal junction (DEJ), while antifibronectin label was retained on loosely dispersed material in the predermal mesenchyme. Dense feather-forming dermis was characterized by loosening of the anti-collagen label along the DEJ, by its spreading throughout the thickness of dermis, and by an overall densification of antifibronectin label. Feather formation coincided with a decrease of anti-collagen and an increase of antifibronectin label density in the dermal feather condensations and in the core of outgrowing feather buds. By contrast, density of anti-collagen-labeled material was highest and anti-fibronectin-labeled material was lowest in interplumar and glabrous skin. In slanting feather buds and feather filaments, distribution of anti-collagen-labeled material exhibited a type-specific cranial-caudal asymmetry. The microheterogeneous distribution of extracellular matrix components might constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of cutaneous appendages.

The role of fibroblasts in dermal vascularization and remodeling of reconstructed human skin after transplantation onto the nude mouse

&NA; The vascularization and the dermal remodeling of two different types of human skin reconstructed “in vitro” and grafted onto the nude mouse were studied. They were composed of human keratinocytes grown either on a human acellular deepidermized dermis (DED), or on a lattice composed of human fibroblasts embedded in bovine type I collagen, a living dermal equivalent (LDE). At different stages after grafting, the transplants were harvested and processed for an immunohistological study with species-specific and non-species-specific antibodies. At one month after grafting, the two types of grafted dermis contained blood vessels whose vascular basement membranes were labeled with a mouse-specific anti-type IV collagen antibody. With an antibody specific for human type IV collagen, a constant labeling of the vascular basement membrane was only observed in the LDE containing fibroblasts. In the DED, a constant association of the mouse endothelial cells with human type IV collagen was observed at early stages after grafting. At later stages, the human type IV collagen progressively disappeared. On the other hand, the dermal-epidermal junction underneath the human epidermis contained human type IV collagen in the two types of reconstructed skin. Labeling with the species-specific antibodies directed against human or murine type I collagen showed that the ratio murine type I collagen versus human type I collagen increased with time, suggesting that the DED is progressively invaded by mouse fibroblasts that produce the mouse collagen. On the other hand, in the LDE, the preexisting bovine

AN IMMUNOHISTOLOGICAL STUDY OF THE REVASCULARIZATION PROCESS IN HUMAN SKIN TRANSPLANTED ONTO THE NUDE MOUSE

The revascularization of human skin transplanted onto the nude mouse was studied by performing double-labeling immunofluorescence microscopy on human skin before transplantation and at different stages, ranging from one week to six months after grafting. With a crossreacting anti-factor-VIII antigen antibody used to identify the endothelial cells, and with human-specific monoclonal antibodies directed against vimentin or HLA-DR antigen, it appeared that the original human endothelial cells of transplanted skin progressively disappear, while murine endothelial cells invade the graft. Moreover, in double-labeling experiments with a crossreacting anti-factor-VIII antibody and a human-specific anti-type-IV-collagen antibody, anastomosis between host and graft vessels and a constant codistribution of graft endothelial cells with human type IV collagen were observed. Finally, double staining with species-specific antibodies directed against murine or human type IV collagen showed that mouse type IV collagen appeared progressively in the graft and was constantly colocalized with human type IV collagen. From these observations, it was concluded that revascularization of human skin transplanted onto the nude mouse proceeds as follows: inoculation; disappearance of human endothelial cells and migration of mouse endothelial cells into the graft over the basement membrane of preexisting human vessels; and production of a new vascular basement membrane by mouse endothelial cells on the original basement membrane of human graft vessels.

Localization and synthesis of type III collagen and fibronectin in human reparative dentine

SummaryThe injury of dental pulp tissue, following caries, is accompanied by the deposit of a typical hard scar tissue known as reparative dentine which should be regarded as the mineralization of a new organic matrix. Highly purified antibodies were used in combination with immunoperoxidase or immunogold technique at the ultrastructural level to reveal the distribution and synthesis of types I and III collagen and fibronectin elaborated by typical matrix-forming cells in the new tissue.Specific immunoperoxidase labelling, on demineralized teeth, clearly demonstrated that type I collagen represents the main type of collagen (88%). It is associated with bundles of fine striated fibrils of type III collagen and in close vicinity with fibronectin and constituted, at least, the new organic matrix of reparative dentine.Immunogold staining gave precise localization mainly over Golgi apparatus for the 3 components, thus suggesting that the cells concerned should not be considered as new odontoblasts but rather as pulpal cells in the process of differentiation participating in the formation of new dentine. Moreover, these events are very similar to those observed during wound healing in other tissues.

Immunolocalization of type IX collagen in normal and spontaneously osteoarthritic canine tibial cartilage and isolated chondrons

OBJECTIVEnThe pericellular localization of type IX collagen in avian and mammalian hyaline cartilages remains controversial, while its distribution during osteoarthritic degeneration is poorly understood. This study aimed to compare and contrast the immunohistochemical distribution of type IX collagen in normal mature and spontaneously osteoarthritic canine tibial cartilage.nnnDESIGNnThick vibratome sectioning techniques were evaluated and compared with isolated chondrons using a range of streptavidin-linked probes in combination with light, confocal and transmission electron microscopy.nnnRESULTSnIn normal intact samples, type IX collagen was concentrated in the pericellular microenvironment, while a weaker extracellular reaction around each chondron separated the territorial matrix from the unstained interterritorial matrix. Further differentiation was evident in isolated chondrons where the fibrous pericellular capsule stained more intensely than the tail and interconnecting segments between columnated chondrons. Two regions of type IX reactivity were identified in osteoarthritic tissue: an intensely stained superficial reactive region below the eroding margins, and normal deep layer cartilage where pericellular staining persists. The superficial reactive region was characterized by chondron swelling and chondrocyte cluster formation, a loss of pericellular type IX staining, and a significant increase in matrix staining between clusters. Disintegration and loss of fibrillar collagens was evident in both the swollen microenvironment and adjacent territorial matrices.nnnCONCLUSIONSnThe results suggest that changes in type IX distribution, expansion of the pericellular microenvironment and chondrocyte proliferation represent key elements in the chondron remodeling and chondrocyte cluster formation associated with osteoarthritic degeneration.

Light and Electron Immunoperoxidase Localization of Minor Disulfide-Bonded Collagens in Fetal Calf Epiphyseal Cartilage

New minor disulfide-bonded collagens were recently described in cartilaginous tissues. The localization of these molecules in epiphyseal proper and growth plate cartilage of fetal calf has been studied using light and electron immunoperoxidase microscopy. The labeling was restricted to the pericellular region of the chondrocytes with an increasing intensity form the superficial to the inner zone of the epiphysis. At the ultrastructural level, the fine-non striated fibrils of the pericellular matrix were stained, demonstrating their collagenous nature. These minor collagenous chains are thus new components of the chondrocyte exoskeleton in which other molecules (proteoglycans, chondronectin, fibronectin and type V collagen) have been previously demonstrated.

Transglutaminase-2: a new endostatin partner in the extracellular matrix of endothelial cells.

Endostatin, a C-terminal fragment of collagen XVIII, binds to TG-2 (transglutaminase-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (Kd=6.8 nM). Enzymatic assays indicated that, in contrast with other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, Arg27 and Arg139, are crucial for its binding to TG-2. They are also involved in the binding to heparin [Sasaki, Larsson, Kreuger, Salmivirta, Claesson-Welsh, Lindahl, Hohenester and Timpl (1999) EMBO J. 18, 6240-6248], and to alpha5beta1 and alphavbeta3 integrins [Faye, Moreau, Chautard, Jetne, Fukai, Ruggiero, Humphries, Olsen and Ricard-Blum (2009) J. Biol. Chem. 284, 22029-22040], suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the hypothesis that the GTP-binding site of TG-2 is a potential binding site for endostatin. Endostatin and TG-2 are co-localized in the extracellular matrix secreted by endothelial cells under hypoxia, which stimulates angiogenesis. This interaction, occurring in a cellular context, might participate in the concerted regulation of angiogenesis and tumorigenesis by the two proteins.