Endogenous tissue engineering: PTH therapy for skeletal repair

Based on its proven anabolic effects on bone in osteoporosis patients, recombinant parathyroid hormone (PTH_1-34) has been evaluated as a potential therapy for skeletal repair. In animals, the effect of PTH_1-34 has been investigated in various skeletal repair models such as fractures, allografting, spinal arthrodesis and distraction osteogenesis. These studies have demonstrated that intermittent PTH_1-34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms, which include effects on mesenchymal stem cells, angiogenesis, chondrogenesis, bone formation and resorption. Furthermore, PTH_1-34 has been shown to enhance bone repair in challenged animal models of aging, inflammatory arthritis and glucocorticoid-induced bone loss. This pre-clinical success has led to off-label clinical use and a number of case reports documenting PTH_1-34 treatment of delayed-unions and non-unions have been published. Although a recently completed phase 2 clinical trial of PTH_1-34 treatment of patients with radius fracture has failed to achieve its primary outcome, largely because of effective healing in the placebo group, several secondary outcomes are statistically significant, highlighting important issues concerning the appropriate patient population for PTH_1-34 therapy in skeletal repair. Here, we review our current knowledge of the effects of PTH_1-34 therapy for bone healing, enumerate several critical unresolved issues (e.g., appropriate dosing regimen and indications) and discuss the long-term potential of this drug as an adjuvant for endogenous tissue engineering.     

Comparison of the expression of cell surface poly-N-acetyllactosamine-type oligosaccharides in PC12 cells with those in its variant PC12D.

To explore the biological role of carbohydrate chains in the process of nerve cell differentiation, we carried out a characterization of the carbohydrate structure of glycoproteins by comparing conventional PC12 cells with variant cells (PC12D). In vitro metabolic labeling of cells with either [(3)H] glucosamine or [(3)H] threonine, together with tomato lectin staining, revealed that nerve growth factor (NGF) stimulation caused a decrease in the poly-N-acetyllactosamine synthesis of high-molecular-weight glycopeptides from PC12 cells. By comparison, the amount of glycopeptides with poly-N-acetyllactosamine from PC12D cells was already significantly low and it was not changed by NGF stimulation. By assaying the glycosyltransferases that participate in poly-N-acetyllactosamine synthesis, the decrease in the amount of the poly-N-acetyllactosamine in PC12D cells as well as NGF-stimulated PC12 cells could be accounted for by a reduction in the activity of poly-N-acetyllactosamine extension enzyme (GnT-i), because the amount of poly-N-acetyllactosamine in both cells precisely correlated with changes in GnT-i activity, whereas the activities of N-acetylglucosaminyltransferase V (GnT-V) and beta 1-4 galactosyltransferase remained unchanged. These results demonstrate that the decrease in poly-N-acetyllactosamine synthesis in PC12 cells occurred prior to neurite formation, whereas PC12D cells were insensitive to this effect. Next, we showed that GnT-i but not GnT-V catalyzed a rate-limiting reaction in the expression of poly-N-acetyllactosamine chains, especially in pheochromocytoma.     

Separation of tubulin-binding and anti-inflammatory activity in colchicine analogs and congeners

The effects of colchicine and its analogs on the carrageenin-induced footpad edema in rats were investigated. The anti-inflammatory effects of colchicine analogs were measured at 3 and 5 hr after the carrageenin injection. Colchicine, 1-demethylcolchicine and 3-demethylcolchicine markedly inhibited the carrageenin edema whereas 2-demethylcolchicine was much less active. Thiocolchicinoids, having a thiomethyl group at C-10 instead of a methoxy group, were considerably less potent. These results suggest that the presence of methoxy groups at C-2 and C-10 in colchicine is necessary to maintain anti-inflammatory activity. Inactivity of deacetylcolchicine indicates that substitution of the amino group at C-7 with electron withdrawing groups is also important. Significant inhibition of carrageenin edema and strong binding to tubulin in vitro were manifested by colchicine, 3-demethylcolchicine, N-butyryldeacetylcolchicine and colchifoline. On the other hand, N-carbethoxydeacetylcolchicine which did bind well to tubulin, did not show much effect on the carrageenin edema. These results suggest that the anti-inflammatory action of colchicinoids may not be regulated through the microtubule system.     

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin)

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

Hyperplastic cellular components of a hemangiopericytoma. An ultrastructural study

Based on ultrastructural features of cellular components of a hemangiopericytoma, hyperplastic cells are classifiable into fibroblast-like (group I), endotheloid (group II) and pericyte-like (group III) cells. The transformation of the group I cells to the group II, or to the group III cells, is pronounced in our electron micrographs and this may imply that the group I cell is the principal cell of origin in this neoplasm. The smooth muscle-like (group IV) cells comprising the media of the arteries and veins in this neoplasm may represent modified, possibly de-differentiated smooth muscle cells reacted to the neoplastic proliferation of the surrounding adventitial (group I) cells.     

Splenic outer periarterial lymphoid sheath (PALS): An immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2-positive macrophages in the rat

Summary In an attempt to reveal the role of antigen-laden marginal metallophil (MM) and other macrophages in the intrasplenic immune response of a specific B-cell lineage to a thymus-independent type-2 antigen (Ficoll conjugated with fluorescein isothiocyanate), simultaneous immuno-histological observations of the involved cells were performed in the rat. By newly established methods of double or triple immunostainings, time-kinetics of the following parameters were studied and compared: (1) the antigen, (2) the specific antibody-forming cells (AFC) directed to the fluorescein-isothiocyanate determinant, (3) proliferating cells labeled with 5-bromo-2-deoxyuridine (BrdU), and (4) macrophage subpopulations recognized by monoclonal antibodies (ED2 and ED3). The antigen localized stably not only in the marginal-zone macrophages but also in the MM except around the follicular area. The increase of BrdU-positive cells was observed from day 2 up to day 4 after antigen injection mostly in the periphery of the periarterial lymphoid sheath (outer PALS), which indicated antigen-induced proliferation. As a novel finding, the majority of AFC, both BrdU-positive and -negative, were either closely associated with the antigen-laden MM, or forming cell clusters with ED2-positive macrophages in the outer PALS. In contrast, there were very few AFC in juxtaposition to antigen-free MM in the follicular area or the antigen-laden marginal zone macrophages. The results led to the proposal of a hypothesis that the antigen-laden MM together with ED2-positive macrophages constitute an immunoproliferative microenvironment for the plasmacellular reaction by accumulating the antigen-specific B-cell lineage and promoting these cells to differentiate into the AFC and to proliferate in the outer PALS.Abbreviations AFC specific antibody-forming cells - BrdU 5-bromo-2-deoxyuridine - Fic-F FITC-conjugated Ficoll - FITC fluorescein isothiocyanate - HRP horseradish peroxidase - MM marginal metallophils - MZ marginal zone - PALS periarterial lymphoid sheath - PBS phosphate-buffered saline - TI2 thymus-independent type-2