Long-term outcomes of an extracorporeal irradiated autograft for limb salvage operations in musculoskeletal tumours: over ten years' observation.

AIMS: We analyzed the long-term outcomes of patients observed over ten years after resection en bloc and reconstruction with extracorporeal irradiated autografts. PATIENTS AND METHODS: This retrospective study included 27 patients who underwent resection en bloc and reimplantation of an extracorporeal irradiated autograft. The mean patient age and follow-up period were 31.7 years (9 to 59) and 16.6 years (10.3 to 24.3), respectively. The most common diagnosis was osteosarcoma (n = 10), followed by chondrosarcoma (n = 6). The femur (n = 13) was the most frequently involved site, followed by the tibia (n = 7). There were inlay grafts in five patients, intercalary grafts in 15 patients, and osteoarticular grafts in seven patients. Functional outcome was evaluated with the Musculoskeletal Tumor Society (MSTS) scoring system. RESULTS: There were no recurrences in the irradiated autograft and the autograft survived in 24 patients (88.9%). Major complications included nonunion (n = 9), subchondral bone collapse (n = 4), and deep infection (n = 4). Although 34 revision procedures were performed, 25 (73.5%) and four (11.8%) of these were performed less than five years and ten years after the initial surgery, respectively. The mean MSTS score at the last follow-up was 84.3% (33% to 100%). CONCLUSION: Considering long-term outcomes, extracorporeal irradiated autograft is an effective method of reconstruction for malignant musculoskeletal tumours Cite this article: Bone Joint J 2019;101-B:1151-1159.

In vitro induction systems for analyses of amphibian organogenesis and body patterning.

The discovery that some well-known growth factors have inducing activity in embryogenesis has accelerated our understanding of embryonic induction. Relevant receptors, signal transduction pathways and patterns of gene expression have been characterized over the past decade. Amphibian embryos have provided an excellent model for analysis of embryonic induction because they are easily surgically manipulated and cultured in vitro, and with the addition of treatment with various inducing factors we have been able to control organogenesis and body patterning during early development in vitro. Activin A, a TGF-beta family protein, has a potent mesoderm-inducing activity on the isolated ectoderm called the animal cap. Activin induces animal caps to differentiate into various mesodermal and endodermal tissues, including beating hearts, in a dose-dependent fashion. Activin, in combination with retinoic acid, also induces the formation of the pronephros, a primitive embryonic kidney. The in vitro induced kidney was confirmed to function in vivo in a transplantation experiment. Furthermore, the activin-induced animal caps organize heads or trunk-and-tails in exactly the same manner as the organizer. The potential use of in vitro induction systems to further our understanding of vertebrate organogenesis and body patterning will be discussed.

In vitro control of organogenesis and body patterning by activin during early amphibian development.

In the process of amphibian development, an embryonic body plan is established through cell division, sequential gene expression, morphogenesis and cell differentiation. The mechanism of body patterning is complex and includes multiple induction events. Activin, a TGF-beta family protein, can induce several kinds of mesodermal and endodermal tissues in animal cap explants in a dose-dependent manner. In a recent study of the role of activin in organogenesis, we succeeded in raising a beating heart by treating animal caps with a high concentration of activin. Activin also participates in kidney organogenesis in combination with retinoic acid. An embryonic kidney induced by activin and retinoic acid in vitro can function in vivo when it is transplanted into a larva in which pronephros rudiments have already been removed. Further, the activin-treated animal caps clearly show organizer actions that are closely related to body patterning along the anteroposterior axis. These experiments will help to serve as a model system for understanding organogenesis and body patterning at the cellular and molecular levels.

Work in progress: the renaissance in amphibian embryology.

Various historical eras in the distant as well as the recent past of amphibian embryology are briefly reviewed. The concepts which emerged from the early years matured, then were laid to rest for several decades. A resurgence, driven by key discoveries with peptide growth factors, and fueled by modern molecular biology methods, is underway. The future for several amphibian research projects should be promising since interest in basic concepts remains strong, and application of frontier methodologies is yielding novel findings.

Cytochalasin B inhibits morphogenetic movement and muscle differentiation of activin-treated ectoderm in Xenopus.

Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin-treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin-treated exp ants. Co-treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin-treated explants.

Role of activin and other peptide growth factors in body patterning in the early amphibian embryo.

The amphibian body plan is established as the result of a series of inductive interactions. During early cleavage stages cells in the vegetal hemisphere induce overlying animal hemisphere cells to form mesoderm. The interaction represents the first major body-patterning event and is mediated by peptide growth factors. Various peptide growth factors have been implicated in mesoderm development, including most notably members of the transforming growth factor-beta superfamily. Identification of the so-called "natural" inducer from among the several candidate peptide growth factors is being achieved by employing several experimental strategies, including the use of a tissue explant assay for testing potential inducers, cloning of marker genes as indices of early induction events, and microinjection of altered peptide growth factor receptors to disrupt normal embryonic inductions. Activin emerges as the most likely choice for assignment of the role of endogenous mesoderm inducer, because it currently best fulfills the rigorous set of criteria expected of such an important embryonic signaling molecule. Activin, however, may not act alone in mesoderm induction. Other peptide growth factors such as fibroblast growth factor might be involved, especially in the regional patterning of the mesoderm. In addition, several genes (e.g., Wnt and noggin), which are expressed after the mesoderm is initially induced, probably assist in further definition of the mesoderm pattern. Following mesoderm induction, the primary embryonic organizer tissue (first described in 1924 by Spemann) develops and contributes further to body patterning by its action as a neural inducer. Peptide growth factors such as activin may also be involved in the inductive event, either directly (by facilitating gene expression) or indirectly (by serving to constrain pathways).

Activation of Stat3 by cytokine receptor gp130 ventralizes Xenopus embryos independent of BMP-4.

Stat3 is one of the main signaling components of cytokine receptors, including gp130. Here we show that activation of cytokine receptor gp130 resulted in a dramatic ventralization of Xenopus embryos and that the ventralization correlated well with Stat3 activation potential of the receptor. This finding led to identification of Xenopus Stat3 (Xstat3), which showed a 95% homology to its murine and human counterparts, at the amino acid level, and was expressed from the one-cell stage throughout development. The mechanism of gp130/XStat3-mediated ventralization proved to be independent of BMP-4. gp130/Xstat3 stimulation inhibited Smad2-induced ectopic axis formation in embryos and Smad2-dependent luciferase activity. A dominant-negative Stat3, in contrast, dorsalized Xenopus embryos, resulting in ectopic axis formation. We propose that Stat3-mediated signaling has the capacity to modify dorsoventral patterning in the early development of Xenopus.

Patterns of gene expression in the core of Spemann's organizer and activin-treated ectoderm in Cynops pyrrhogaster.

The presumptive pharyngeal endoderm region of the Cynops early gastrula induces head or trunk-tail structures in sandwich culture. Activin-treated ectoderm can mimic this phenomenon at least at the histological level. The patterns of expression of organizer-specific genes were examined to compare these two inductive materials at the molecular level. A chordin cDNA clone from Cynops pyrrhogaster (Cychd) was isolated by reverse transcription-polymerase chain reaction (RT-PCR). Cychd mRNA was first detected in the presumptive pharyngeal endoderm and prechordal plate regions of stage 11 embryos, and was expressed continuously until stage 20. The spatiotemporal expression pattern of Cychd was similar to that of Xenopus chordin. The patterns of expression of organizer-related genes in the pharyngeal endoderm and activin-treated ectoderm were compared by RT-PCR analysis. Expression of Cychd in these two materials peaked at the time when they can induce head structures in sandwich culture. Expression of fork head and goosecoid did not change in the presumptive pharyngeal endoderm over this period. Cychd may play a key role in head formation in the Cynops embryo.

Activin-treated ectoderm has complete organizing center activity in Cynops embryos.

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae-early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk-rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin-treated ectoderm. These findings show that activin-treated ectoderm can act as the complete organizing center in Cynops.

Activin treated urodele ectoderm: a model experimental system for cardiogenesis.

The tissue interactions which comprise the inductive phenomena associated with urodele heart morphogenesis are relatively well understood. In order to take full advantage of the experimental potential of this system formulation of an in vitro tissue culture system would be very helpful. Herein are described conditions for culturing Cynops pyrrhogaster early gastrula ectoderm tissue in the presence of the peptide growth factor activin. Two-week old explant cultures frequently displayed beating heart-like rudiments within. The beating frequency was measured and the extent to which cytodifferentiation mimicked normal heart differentiation assessed. Both measurements provided optimistic assessments which should encourage further exploitation of this model system.

Control of the embryonic body plan by activin during amphibian development.

Embryonic induction plays an important role in establishing the fundamental body plan during early amphibian development. The factors mediating this embryonic induction have, however, only recently been discovered. In the mid-1980's, certain peptide growth factors belonging to the FGF and TGF-beta families were found to have a mesoderm-inducing effect on isolated Xenopus blastula ectoderm. The study of embryonic induction subsequently expanded rapidly and knowledge at the molecular level has gradually accumulated. One of these peptide growth factors, activin, a member of the TGF-beta superfamily, is present maternally in the Xenopus early embryo and induces various mesodermal and endodermal tissues in isolated presumptive ectoderm. After exposure of presumptive ectoderm to activin, many genes are expressed in the same manner as in normal embryogenesis. Ectoderm treated with activin can induce a complete secondary embryo, the same as the organizer does in transplantation experiments. These findings suggest that activin is one of the first induction signals responsible for establishing the embryonic body plan in early amphibian development. In this article we shall review to what extent we can control the embryonic body plan in vitro, referring to some significant findings in this field.

Head and trunk-tail organizing effects of the gastrula ectoderm of Cynops pyrrhogaster after treatment with activin A.

Differentiation tendency and the inducing ability of the presumptive ectoderm of newt early gastrulae were examined after treatment with activin A at a high concentration (100 ng/ml). The activin-treated ectoderm differentiated preferentially into yolk-rich endodermal cells. Combination explants consisting of three pieces of activin-treated ectoderm formed neural tissues and axial mesoderm along with endodermal cells. However, the neural tissue was poorly organized and never showed any central nervous system characteristics. When the activin-treated ectoderm was sandwiched between two sheets of untreated ectoderm, the sandwich explants differentiated into trunk-tail or head structures depending on the duration of preculture of activin-treated ectoderm in Holtfreter's solution. Short-term (0-5 h) precultured ectoderm induced trunk-tail structures accompanied by axial organs, alimentary canal and beating heart. The arrangement of the explant tissues and organs was similar to that of normal embryos. However, archencephalic structures, such as forebrain and eye, were lacking or deficient. On the other hand, long-term (10-25 h) precultured ectoderm induced archencephalic structures in addition to axial organs. Lineage analysis of the sandwich explants using fluorescent dyes revealed that the activin-treated ectoderm mainly differentiated into endodermal cells and induced axial mesoderm and central nervous system in the untreated ectoderm. These results suggest that activin A is one of the substances involved in triggering endodermal differentiation and that the presumptive ectoderm induced to form endoderm displays "trunk-tail organizer" or "head organizer" effects, depending on the duration of preculture.

Isolation and characterization of native activin B.

To examine whether activin binds to follistatin, an activin-binding protein, to form a complex in vivo, we attempted to purify activin-follistatin complex from porcine follicular fluid. Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B are present as a complex with follistatin in the follicular fluid. Reverse-phase high performance liquid chromatography of the purified complex yielded follistatin and activins A, AB, and B. The activity of the purified activin B was found to be significantly lower than those of other activins in various assay systems such as stimulation of follicle-stimulating hormone secretion, induction of erythrodifferentiation, and potentiation of expression of gonadotropin receptors on ovarian cells. Moreover, binding of 125I-activin A to erythroleukemic cells which are activin-responsive was competed by activin B with approximately 10-fold lower potency compared with other activins. In contrast to these results, activin B was proved to have a potent Xenopus mesoderm-inducing activity, comparable with that of other activins. This indicates that, unlike activins A and AB, activin B can only elicit mesoderm-inducing activity and cannot function in other biological systems, suggesting a specific role of activin B in early development and unknown biological functions.

[Angiographic studies of atherosclerotic changes in coronary vein grafts after operation].

A total of 230 vein grafts were studied angiographically in 116 unselected survivors of 260 coronary bypass operations performed from May 1977 through October 1989 in order to investigate atherosclerotic changes in coronary vein grafts after operation. These patients were divided into three groups according to the interval from operation to angiography. In group A (30 patients) the interval was less than one year (mean interval 8.2 months), in group B (73 patients) from one to five years (mean interval 19.2 months) and in group C (13 patients) more than 5 years (mean interval 96.6 months). Fifty-five vein grafts were in group A, 153 vein grafts in group B and 22 vein grafts in group C. The graft patency rate of each group was 83.6%, 89.5% and 90.9% respectively (N.S.). To classify angiographic appearances we believe to be caused by atherosclerosis, we devised a grading system. Category I indicated that the graft outline was completely smooth without any irregularity; Category II indicated that less than 50% of the estimated surface area of the graft intima was irregular; Category III indicated that more than 50% of the intima was involved. Significant stenosis indicated narrowing reducing the lumen to less than 50% of the graft. Of the 203 patent grafts 181 grafts (89%) were in Category I, 22 grafts (11%) in Category II, but no graft in Category III. In group A of the 46 patent grafts 45 grafts (98%) were classified as Category I and 1 graft (2%) was classified as Category II.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose and time-dependent mesoderm induction and outgrowth formation by activin A in Xenopus laevis.

We examined the quality of mesoderm induced by the action of activin A on the Xenopus presumptive ectoderm when various concentrations and treatment times were employed. The minimum concentration of activin A to induce mesodermal tissues was inversely proportional to its treatment time. The explants differentiated into different types of mesodermal tissues, from ventral-type to dorsal-type depending on the concentration of activin A and its treatment time. To confirm whether activin A has a role in establishing axial organization, activin A was injected into the blastocoel of late blastulae. About 70% of the injected embryos formed secondary tail-shaped outgrowths in which muscle and neural tube differentiated. The amount of activin A to form secondary outgrowths was 0.5-2.5 pg, roughly consistent with the amount estimated from in vitro experiments. As we have detected almost the same amount of activin homologue in the early embryos (Asashima et al., 1991a), we speculate that activin A may be the natural mesodermal inducer, and that it is responsible for establishing axial organization in the Xenopus embryo.

Preparation of serum albumin microcapsules.

Simple coacervation of bovine serum albumin was studied to prepare biodegradable microcapsules. Three types of microcapsules, which differed in shape, were obtained by changing either core size or the bovine serum albumin concentrations of the coacervating systems. Mononuclear microcapsules were prepared by using spherical poly(acrylonitrile) beads as a core material.