Small unilamellar liposomes from mixed natural and polymeric phospholipids: stability and susceptibility to phospholipase A2.

The concept of the uncorkable liposome composed of phase-separated mixtures of a polymerized phospholipid and an enzymically digestible phospholipid has been investigated, using small unilamellar vesicles composed of mixtures of (polymerized) dienoylphosphatidylcholine (DENPC) and dimyristoylphosphatidylcholine (DMPC). Mixed liposomes, even those containing only 10% DENPC, were much more stable than DMPC liposomes, as indicated by the release of entrapped [3H]inulin or [14C]glucose. DMPC liposomes released entrapped solute on exposure to phospholipase A2, whereas mixed vesicles were resistant. The results are compared with those of an earlier study on monolayers of similar compositions. It is concluded that the liposomes, like the monolayers, are phase-mixed, and that uncorkable liposomes cannot be constructed from the phospholipid mixture employed. It is proposed that, until further experimental evidence is produced, the enzymatically uncorkable liposome must be regarded as a theoretical construct.

The effect of exogenous transforming growth factor-beta 2 on healing fractures in the rabbit.

The effects of exogenous TGF-beta 2 on normally healing fractures were investigated to see if healing can be accelerated; TGF-beta s stimulate bone and cartilage formation on calvariae and long bones. TGF-beta 2 (60 or 600 ng) was injected around the developing callus of rabbit tibial fractures healing under stable, or unstable, mechanical conditions 4 days after fracture. The fractures were examined 5, 7, 10, and 14 days after fracture. A large amount of edema developed around the injection sites. The callus of fractures healing under stable mechanical conditions consists almost entirely of bone. The effects of 60-ng injections of TGF-beta 2 are minimal, but 600-ng doses lead to a small increase in the size of the callus. The callus of fractures healing under unstable mechanical conditions has a large area of cartilage over the fracture site with bone on each side. The effects of TGF-beta 2 on unstable fractures are to retard and reduce bone and cartilage formation in the callus. The overall size of the callus is not affected. In conclusion, TGF-beta 2 does not stimulate fracture healing under either stable, or unstable, mechanical conditions during the initial healing phase. It is argued that agents which stimulate bone formation can retard remodeling when treatment is extended into the remodeling phase of healing.

Radiolabeling, stability, and body distribution in rats, of low molecular weight polylactide homopolymer and polylactide-polyethyleneglycol copolymer.

In order to study its fate in vivo, a low molecular-weight polylactide homopolymer was derivatized with a p-methoxyphenyl moiety, so as to make it susceptible to radiolabeling with 125I. A low molecular weight polylactide-polyethyleneglycol copolymer capped with ap-methoxyphenyl residue was also synthesized. The derivatized polymers were successfully [125I]iodinated in organic medium. The radiolabeled products were freed from [125I]iodide by dialysis and shown to be stable for 24 h on incubation at 37 degrees C in buffered saline or in blood. On longer incubation at 37 degrees C in buffered saline the radiolabeled polylactide released [125I]iodide and [125I]iodinated 3-(p-methoxyphenyl)propionic acid. The radiolabeled copolymer was more stable on incubation at 37 degrees C in buffered saline, but some [125I]iodide was released. The tissue distribution of radioactivity was determined 5 min, 1, 5 and 24 h after injecting male rats with 125I-labeled homopolymer or copolymer. Intravenous, intraperitoneal and subcutaneous injection routes were employed. Further rats were injected with [125I]iodide, to aid interpretation of the data. After administration of labeled homopolymer, a high concentration of radioactivity was found in the liver tissue. The levels slowly decreased over 24 h, and the polymer was successively found in the small and large intestine and the faeces. This is probably indicative of excretion via the bile. Concurrently radioactivity was excreted in the urine. After administration of labeled copolymer, a high concentration of radioactivity was found in the liver and the residual soft tissue, the latter fraction containing two-thirds of the radioactivity one hour after injection. The precise tissue location that this result indicates was not identified. After 1 h radioactivity was excreted in the faeces, again probably via the bile, and in the urine. Tissue distributions after intraperitoneal or subcutaneous injections were concordant with the above results and interpretations, with the additional factor of slow clearance from the injection site.

Molecular failure of apoptosis: inappropriate cell survival and mutagenesis?

Since cell death by apoptosis is achieved through complex interactions between numerous molecular components, cells may fail to die when stimulated because of molecular abnormalities in the apoptosis pathway or in its control mechanisms. Such inappropriate cell survival is well established when apoptosis is suppressed by elevated expression of bcl-2, at least for some cell types. Many cells undergo apoptosis at moderate levels of DNA damage and suppression of such apoptosis might be expected to increase the rate of mutation because of the persistence of cells with damaged DNA. We and others have now confirmed this prediction in bcl-2 transfected cells. Suppression of the apoptosis pathway can only lead to inappropriate cell survival if it relates to events before the cell becomes committed to die. We have analyzed this question for agents that inhibit the caspases, the site-specific proteases which form the biochemical core of the process of apoptosis. We have shown that inhibition of certain caspases does lead to the survival of Jurkat human T-cells induced to undergo Fas-mediated apoptosis.

Immunolocalization of basement membrane components and beta 1 integrin in the chick wing bud identifies specialized properties of the apical ectodermal ridge.

To examine whether the extracellular matrix (ECM) plays a role in mediating interactions between the apical ectodermal ridge (AER) and the subjacent mesoderm in the chick wing bud, we used immunohistochemistry to locate the following tissue components during wing morphogenesis: types I and IV collagens, fibronectin, the basal lamina form of heparan sulphate proteoglycan (HSPG), laminin, and the beta 1 integrin subunit. The notch region at the base of the AER exhibits particularly strong labelling for type IV collagen, fibronectin, laminin, and beta 1 integrin. This suggests that the ridge cells are firmly anchored to their underlying basement membrane. In nonridge ectoderm, the beta 1 integrin subunit is present only at the basal cell surface, whereas in the AER it has a pericellular distribution. The localization of beta 1 integrin receptors at the lateral ridge cell surfaces, in the apparent absence of fibronectin, collagens I and IV, and laminin, suggests that they may function in cell-cell adhesion in the AER. The normal AER-mesenchyme interface was compared to an experimental situation in which the AER flattens. This was induced in the anterior region of the wing bud by inserting an impermeable barrier at intersomite level 17/18, at stage 21. At 12 hr (stage 23) and 24 hr (stage 25) after the operation, each of the ECM components listed above is uniformly distributed along the experimental epithelial-mesenchymal interface. By 24 hr postoperation, the beta 1 integrin subunit is restricted to the basal surface of the flattened apical ectoderm. Similar changes occur in the AER as it flattens during later stages of normal development. These results point to a possible role for the ECM and integrin receptors in maintaining the thickened structure of the AER.

Digoxigenin as a probe label for in situ hybridization on skeletal tissues.

Non-specific staining was encountered using digoxigenin-labelled cDNA probes for in situ hybridization on sections of skeletal tissues. This staining was most pronounced in cartilaginous matrices. Experimental procedures indicate that the background staining is caused by antibody-binding to hydrophobic sites in the tissues revealed by proteolytic permeabilization. A protocol for minimizing this background is described.

Exogenous fibroblast growth factors-1 and -2 do not accelerate fracture healing in the rabbit.

Both fibroblast growth factors-1 (acidic FGF) and -2 (basic FGF) increase the proliferation of osteoblasts and chondrocytes in vitro and FGF-2 stimulates angiogenesis and bone formation in vivo. To test their effects on rabbit tibial fracture-healing under stable and unstable mechanical conditions, 3 micrograms of either FGF-1 or FGF-2 was injected around rabbit tibial fractures on day 4 after fracture. Neither growth factor had a significant effect on either the size of, or the amounts of bone and cartilage in, the 10-day callus irrespective of the mechanical conditions under which the fracture was healing. The 10-day FGF-2-treated calluses were, however, more mature than FGF-1-treated calluses because the cartilage was separated from the periosteum by bone and endochondral ossification had progressed further. In conclusion, the application of FGF-1 or FGF-2 to normally healing fractures of the rabbit tibia does not have a significant effect on the rate of healing.

The expression of collagen mRNAs in normally developing neonatal rabbit long bones and after treatment of neonatal and adult rabbit tibiae with transforming growth factor-beta 2.

Normal transverse growth of long bones is by periosteal appositional bone formation, balanced by endosteal resorption. Changes in the distribution of cells that are expressing collagen mRNAs during growth were determined using digoxigenin-labelled riboprobes. In neonatal rabbit tibiae osteoblasts expressing type I collagen mRNA are found on periosteal, and at early stages on endosteal, bone surfaces and lining peripheral cavities. Occasional osteocytes express type I collagen mRNA very weakly. The pattern is disrupted when transforming growth factor-beta 2 (TGF-beta 2) is injected daily into the periosteum of neonatal animals; there is increased bone, and later cartilage, formation. Three injections of 20 ng TGF-beta 2 onto the tibia of 3-day-old rabbits led to an increase of periosteal osteoblasts that express the mRNA for type I collagen. Some endosteal osteoblasts and osteocytes in newly-formed peripheral woven bone also express the mRNA. After five injections chondrocytes expressing type II collagen mRNA are found around the injection site. Similar injections of TGF-beta 2 in old rabbits induce only fibrous tissue within which some cells express type I collagen mRNA. This precise localization of mRNAs shows that the expression of type I or II collagen mRNA is here restricted to osteoblasts and chondrocytes, respectively.

The effects of age on the response of rabbit periosteal osteoprogenitor cells to exogenous transforming growth factor-beta 2.

Additional bone and cartilage are formed if transforming growth factor-beta is injected into the periosteum of calvariae or long bones. To investigate this further, transforming growth factor-beta 2 was injected into the periosteum of the tibia of 3-day-old, 3-month-old and 2-year-old rabbits. In all instances, there was an increase in proliferation of the cells of the cambial layer of the periosteum, that is, the osteoprogenitor cells, and breakdown of the fibrous layer. Oedema was induced in the surrounding connective tissues. Over the experimental period the normal neonatal tibia is undergoing rapid growth; there is periosteal bone formation and endosteal resorption. In the experimental neonatal tibiae, an increase in periosteal bone formation is seen after three injections of 20 ng of transforming growth factor-beta 2, which is accompanied by cartilage after five injections; the amounts of induced bone and cartilage increase with the number of injections. The chondrocytes hypertrophy after 4 days and the cartilage is replaced by bone endochondrally. In contrast, after seven injections of 20 ng transforming growth factor-beta 2, there is only a small amount of new bone on the 3-month-old tibia and none on the 2-year-old tibia. One day after seven injections of 200 ng transforming growth factor-beta 2, there is a small amount of bone formation, while seven days after cartilage is found as small discrete nodules on the 3-month-old tibia, but as small areas within the bone on the 2-year-old tibia. It is concluded that the primary effect of transforming growth factor-beta 2 in this experimental model is to increase the proliferative rate of the osteoprogenitor cells in the periosteum. It is argued that transforming growth factor-beta 2 does not initiate osteoblastic or chondrocytic differentiation of osteoprogenitor cells. It is suggested that their differentiation is controlled by the local environment, in particular, the vascularity and locally circulating growth factors.

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells.

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA. We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.

Experimental analysis of the role of ECM in the patterning of the distal tendons of the developing limb bud.

We have shown previously that from stage 27 the distal growing region of the limb exhibits a tenascin-rich sheet of extracellular matrix termed the "mesenchyme lamina" (ML), which runs from the ectodermal basement membrane in a proximal direction until it contacts the distal tip of the muscle blocks. This study reports experimental evidence that the mesenchyme lamina is a pretendinous structure that controls the spatial organization of the flexor and extensor tendons of the distal part of the chick leg. Two sets of experiments were designed to alter the ML position and examine subsequent tendon pattern formation. In a first series of experiments limbs with digits lacking phalangeal elements were induced by AER removal at stages 26 and 27. This procedure induced an abnormal arrangement of the ML around the distal tip of each terminal phalange of the truncated digit, which was followed by the development of a precisely similar pattern of abnormal extensor and flexor tendons. In the second set of experiments, an extradigit was induced to form in the interdigital mesenchyme through surgical removal of the marginal ectoderm of the third interdigit of stage 29 leg buds. By day 4 post-operation, a chondrogenic extradigit had formed, together with a ML that ran from the cartilage to the normal ventral flexor and dorsal extensor tendons. By day 6 post-operation, the experimentally induced ML had transformed into a tendinous structure connecting with the adjacent normal tendon. Both experiments show that the position of the ML defines the position of subsequent tendon development, thus supporting its role as a pretendinous structure which might be responsible for the alignment of the pretendinous condensing cells.

The extracellular matrix architecture relating to myotendinous pattern formation in the distal part of the developing chick limb: an ultrastructural, histochemical and immunocytochemical analysis.

In the later developmental stages (Hamburger and Hamilton, 25-34) the distal part of the chick leg possesses a distinctive extracellular matrix (ECM) architecture which relates to myotendinous patterning. There are two components: firstly, a system of dorsoventrally oriented fibrils which link the two ectodermal surfaces through the undifferentiated distal mesenchyme and secondly, a 'mesenchyme lamina' originates at the basement membrane distally, but proximally runs through the mesoderm, subjacent and parallel to the basement membrane. The 'mesenchyme lamina' appears to be a precursor of developing tendons and is spatially related to the distal tips of the myogenic blocks. As developing tendons form on the inner surface of the lamina at its proximal end, it becomes less distinct and disappears. Further dorsoventral fibrils run from the 'mesenchyme lamina' into the developing condensations and chondrogenic elements of the phalanges. The architecture of the ECM was revealed by silver and lectin staining (peanut and Ricinus communis agglutinins, PNA and RCA I), by immunocytochemistry (for fibronectin, tenascin, collagen type I) and by ultrastructural analysis. Both components stain with silver, PNA following neuraminidase digestion, RCA I, tenascin and collagen type I. However, the dorsoventral fibrils are positive for fibronectin and negative for PNA, while conversely the mesenchyme lamina is positive for PNA but much less so for fibronectin. Tenascin has been shown to be a specialized mesenchyme component of tendons and myotendinous junctions (Chiquet and Fambrough, 1984). Such a basement membrane forming a 'mesenchyme lamina' appears to be unique in epithelial-mesenchymal developing systems and points to an ectodermal role in tendon pattern formation within the mesenchyme. We discuss the possible role of mechanical force in converting the dorsoventral tenascin-positive fibrils into the localized pattern of tendon insertions into the proximal parts of the phalanges. Distally the dorsoventral fibrils may shape the digital plate by pulling together the two ectodermal surfaces. A similar ECM architecture is found in corresponding stages in the developing wing.