Surgical timing after chemoradiotherapy for rectal cancer, analysis of technique (STARRCAT): results of a feasibility multi-centre randomized controlled trial.

BACKGROUND: The optimal time of rectal resection after long-course chemoradiotherapy (CRT) remains unclear. A feasibility study was undertaken for a multi-centre randomized controlled trial evaluating the impact of the interval after chemoradiotherapy on the technical complexity of surgery. METHODS: Patients with rectal cancer were randomized to either a 6- or 12-week interval between CRT and surgery between June 2012 and May 2014 (ISRCTN registration number: 88843062). For blinded technical complexity assessment, the Observational Clinical Human Reliability Analysis technique was used to quantify technical errors enacted within video recordings of operations. Other measured outcomes included resection completeness, specimen quality, radiological down-staging, tumour cell density down-staging and surgeon-reported technical complexity. RESULTS: Thirty-one patients were enrolled: 15 were randomized to 6 and 16-12 weeks across 7 centres. Fewer eligible patients were identified than had been predicted. Of 23 patients who underwent resection, mean 12.3 errors were observed per case at 6 weeks vs. 10.7 at 12 weeks (p = 0.401). Other measured outcomes were similar between groups. CONCLUSIONS: The feasibility of measurement of operative performance of rectal cancer surgery as an endpoint was confirmed in this exploratory study. Recruitment of sufficient numbers of patients represented a challenge, and a proportion of patients did not proceed to resection surgery. These results suggest that interval after CRT may not substantially impact upon surgical technical performance.

Evaluating the role of fluorodeoxyglucose positron emission tomography-computed tomography in multi-disciplinary team recommendations for oesophago-gastric cancer.

BACKGROUND: National guidelines recommend that fluorodeoxyglucose positron emission tomography-computed tomography (PET-CT) is performed in all patients being considered for radical treatment of oesophageal or oesophago-gastric cancer without computerised tomography scan (CTS) evidence of metastasis. Guidance also mandates that all patients with cancer have treatment decisions made within the context of a multi-disciplinary team (MDT) meeting. Little is known, however, about the influence of PET-CT on decision making within MDTs. The aim of this study was to assess the role of PET-CT in oesophago-gastric cancer on MDT decision making. METHODS: A retrospective analysis of a prospectively held database of all patients with biopsy-proven oesophageal or oesophago-gastric cancer discussed by a specialist MDT was interrogated. Patients selected for radical treatment without CTS evidence of M1 disease were identified. The influence of PET-CT on MDT decision making was examined by establishing whether the PET-CT confirmed CTS findings of M0 disease (and did not change the patient staging pathway) or whether the PET-CT changed the pathway by showing unsuspected M1 disease, refuting CTS suspicious metastases, or identifying another lesion (needing further investigation). RESULTS: In 102 MDT meetings, 418 patients were discussed, of whom 240 were initially considered for radical treatment and 238 undergoing PET-CT. The PET-CT confirmed CTS findings for 147 (61.8%) and changed MDT recommendations in 91 patients (38.2%) by (i) identifying M1 disease (n=43), (ii) refuting CTS suspicions of M1 disease (n=25), and (iii) identifying new lesions required for investigations (n=23). CONCLUSION: The addition of PET-CT to standard staging for oesophageal cancer led to changes in MDT recommendations in 93 (38.2%) patients, improving patient selection for radical treatment. The validity of the proposed methods for evaluating PET-CT on MDT decision making requires more work in other centres and teams.

Developmental plasticity of human foetal femur-derived cells in pellet culture: self assembly of an osteoid shell around a cartilaginous core.

This study has examined the osteogenic and chondrogenic differentiation of human foetal femur-derived cells in 3-dimensional pellet cultures. After culture for 21-28 days in osteogenic media, the pellets acquired a unique configuration that consisted of an outer fibrous layer, an osteoid-like shell surrounding a cellular and cartilaginous region. This configuration is typical to the cross section of the foetal femurs at the same age and was not observed in pellets derived from adult human bone marrow stromal cells. Time course study showed that after 7-14 days, the cells of the inner cellular region were viable, proliferated rapidly, and were immuno-positive for c-myc, as well as for bone sialoprotein and type I collagen. After 21-28 days, the cells accumulated at the inner edge of the osteoid shell. The direction of osteoid formation thus differed from that of periosteal bone formation. Following micro-dissection of the human foetal femurs into epiphyses, bone cylinder and hypertrophic cartilage, epiphyseal chondrocytes and osteoblasts both gave rise to osteoid-shell forming cells. These studies demonstrate the developmental plasticity of human foetal skeletal and epiphyseal chondrocytes and suggest that the microenvironment modulates lineage commitment and matrix formation. Furthermore, this ex vivo model offers a new approach to delineate human bone development as well as a model with potential application for evaluation of therapeutic compounds for bone formation.

Pathobiology of osteoarthritis: pathomechanisms and potential therapeutic targets.

Osteoarthritis, a degenerative joint disease, is the most disabling condition of the Western world. It affects first and foremost the articular cartilages and leads to a molecular and supramolecular destruction of the extracellular cartilage matrix. In addition, the cells, the chondrocytes, show severe alterations of their phenotype: they get anabolically and catabolically activated, change accordingly their gene expression pattern, lose their differentiated phenotype, and undergo focally cell death and cell degeneration. All these processes represent potential targets for therapeutic intervention and drug development. Apart from the cartilage itself, however, other joint tissues are also involved in the disease: thus, the synovial capsule and membrane as well as the subchondral bone account not only for most of the symptoms of the disease, but are also presumably involved in the progression of the degenerative process. Both, inflammation and stiffening within the joint capsule accelerate joint destruction. Stiffening of the subchondral bone increases the mechanical stress over the overlying cartilage during physiological movement. Altogether, there is a plethora of tissues, disease processes and targets for treating osteoarthritic joint degeneration, which will need to be followed up systematically in the future.

DNA methylation in osteoarthritic chondrocytes: a new molecular target.

OBJECTIVE: To review the current knowledge of the mechanism of DNA methylation, its association with transcriptional silencing, possible mechanisms of hyper- and hypomethylation and how epigenetic changes may relate to the pathogenesis of osteoarthritis (OA). METHODS: Journal literature was searched using Pubmed. Since there are very few publications directly on epigenetic phenomena in OA, the search was extended to give an overview of epigenetic mechanisms as they relate to the molecular mechanisms of the disease. RESULTS: While the epigenetics of cancer cells have been intensively investigated, little attention has so far been paid as to whether epigenetic changes contribute to the pathology of non-neoplastic diseases such as OA. This review explains the mechanisms of DNA methylation, its role in transcriptional regulation, and possible demethylation mechanisms that may be applicable to OA. Preliminary evidence suggests that changes in DNA methylation, together with cytokines, growth factors and changes in matrix composition, are likely to be important in determining the complex gene expression patterns that are observed in osteoarthritic chondrocytes. CONCLUSION: Early evidence points to a role of epigenetics in the pathogenesis of OA. Since epigenetic changes, although heritable at the cellular level, are potentially reversible, epigenetics could be a new molecular target for therapeutic intervention, especially early in the disease.

MMP-9/gelatinase B is a gene product of human adult articular chondrocytes and increased in osteoarthritic cartilage.

OBJECTIVE: Collagen fibril degeneration involves initially the cleavage within the triple helix by the collagenases 1 (MMP-1) and 3 (MMP-13), but then mainly involves also the gelatinases A (MMP-2) and B (MMP-9). The objective of this study was to determine the quantitative expression levels as well as the distribution in normal and osteoarthritic cartilage of gelatinase B and in cultured articular chondrocytes with and without stimulation by Il-1Beta. METHODS: Conventional and real-time quantitative PCR technology and immunohistochemistry were used to determine gelatinase B expression on the mRNA and protein level. RESULTS: Conventional PCR analysis could demonstrate the presence of gelatinase B mRNA only in osteoarthritic chondrocytes. Real-time quantitative PCR confirmed the increased expression of gelatinase B mRNA expression in osteoarthritic chondrocytes. No significant up-regulation of gelatinase B was observed by Il-1Beta. Immunostaining for gelatinase B showed the presence of gelatinase B in a subset of normal and in a large portion of osteoarthritic chondrocytes with a more extended distribution in the latter. CONCLUSION: In osteoarthritic cartilage destruction, gelatinase B is involved in collagen destruction though still at a very much lower level than gelatinase A. Only a very small subset of normal adult articular chondrocytes express gelatinase B in vivo suggesting that gelatinase B unlike gelatinase A is hardly or only very focally involved in physiological collagen turnover.

Changes in immunolocalisation of beta-dystroglycan and specific degradative enzymes in the osteoarthritic synovium.

OBJECTIVE: To investigate the immunolocalisation of beta-dystroglycan (beta-DG) and specific matrix metalloproteinases (MMPs)-3, -9, -13 and a disintegrin like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) within the joint tissues of patients with osteoarthritis (OA) and unaffected controls. DESIGN: Cartilage, synovium and synovial fluid were obtained from the hip joints of five osteoarthritic (patients undergoing total hip replacement) and five control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for beta-DG protein using Western blot technique and by immunohistochemistry for tissue distribution of beta-DG, MMP-3, -9, -13, and ADAMTS-4. RESULTS: beta-DG was detected in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Importantly, beta-DG was detected in endothelium of blood vessels of OA synovium, but not in the control endothelium. In the endothelium of osteoarthritic synovial blood vessels, beta-DG co-localised with MMP-3 and -9. MMP-13 and ADAMTS-4 showed no endothelial staining, and only weak staining of the vascular smooth muscle was found. In contrast, we did not detect beta-DG protein in cartilage or synovial fluid. CONCLUSIONS: beta-DG has been shown to have a role in angiogenesis, and our results demonstrate for the first time that there are clear differences in beta-DG staining between OA and control synovial blood vessels. The specific immunolocalisation of beta-DG within endothelium of inflamed OA blood vessels and its co-localisation with MMP-3 and -9, reported to have pro-angiogenic roles and believed to be involved in beta-DG cleavage, may also suggest that beta-DG plays a role in angiogenesis accompanying OA.

Osteoarthritis: pathobiology-targets and ways for therapeutic intervention.

Osteoarthritis is first and foremost the ongoing destruction of the articular cartilages of joints. Therefore, the extracellular matrix and the cells of the articular cartilages are the primary targets of osteoarthritis therapy. This tries to inhibit enzymatic destruction of the extracellular cartilage matrix as well as the modification of the cellular phenotype of the chondrocytes: cell degeneration and cell death are alongside anabolic activation and stabilization of the cellular phenotype of major interest. However, apart from the cartilage and its cells, other tissues of the joints are also important for the symptoms of the disease, which basically all originate outside the articular cartilage. In addition, changes in the subchondral bone as well as the synovial capsule and membrane are important at least for the progression of the disease process. All the named tissues offer different directions and ways for therapeutic intervention.

Life-threatening hematuria requiring transcatheter embolization following radiofrequency ablation of renal cell carcinoma.

Radiofrequency ablation is increasingly being acknowledged as a valid treatment for renal cell carcinoma in patients in whom definitive curative resection is deemed either undesirable or unsafe. A number of published series have shown the technique to have encouraging results and relatively low complication rates. In this article, we report a case of delayed life-threatening hematuria requiring transcatheter embolization of a bleeding intrarenal artery in a patient who had undergone imaging-guided radiofrequency ablation of a 3 cm renal cell carcinoma. To our knowledge, such a complication has not been reported previously.

Chondroptosis: a variant of apoptotic cell death in chondrocytes?

Evidence has accumulated in recent years that programmed cell death (PCD) is not necessarily synonymous with the classical apoptosis, as defined by Kerr and Wyllie, but that cells use a variety of pathways to undergo cell death, which are reflected by different morphologies. Although chondrocytes with the hallmark features of classical apoptosis have been demonstrated in culture, such cells are extremely rare in vivo. The present review focuses on the morphological differences between dying chondrocytes and classical apoptotic cells. We propose the term 'chondroptosis' to reflect the fact that such cells are undergoing apoptosis in a non-classical manner that appears to be typical of programmed chondrocyte death in vivo. Unlike classical apoptosis, chondroptosis involves an initial increase in the endoplasmic reticulum and Golgi apparatus, reflecting an increase in protein synthesis. The increased ER membranes also segment the cytoplasm and provide compartments within which cytoplasm and organelles are digested. In addition, destruction occurs within autophagic vacuoles and cell remnants are blebbed into the lacunae. Together these processes lead to complete self-destruction of the chondrocyte as evidenced by the presence of empty lacunae. It is speculated that the endoplasmic reticulum pathway of apoptosis plays a greater role in chondroptosis than receptor-mediated or mitochondrial pathways and that lysosomal proteases are at least as important as caspases. Because chondroptosis does not depend on phagocytosis, it may be more advantageous in vivo, where chondrocytes are isolated within their lacunae. At present the initiation factors or the molecular pathways involved in chondroptosis remain unclear.

Novel osteoinductive biomimetic scaffolds stimulate human osteoprogenitor activity--implications for skeletal repair.

The development of new bone formation strategies offers tremendous therapeutic implications in a variety of musculoskeletal diseases. One approach involves harnessing the regenerative capacity of osteoprogenitor bone cells in combination with biomimetic scaffolds generated from appropriate scaffold matrices and osteoinductive factors. The aims of our study were to test the efficacy of two innovative osteoinductive agents: the osteoblast stimulating factor-1 (osf-1), an extracellular matrix-associated protein, and osteoinductive extracts of Saos-2 cells on human osteoprogenitor cells. Saos-2 extracted osteoinductive factors significantly stimulated alkaline phosphatase specific activity in basal and osteogenic conditions. Osf-1 significantly stimulated chemotaxis, total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase specific activity at concentrations as low as 10 pg/ml compared with control cultures. Osteoinductive factors present in Saos-2 cell extracts and osf-1 promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on 3-D scaffolds. The successful generation of 3-D biomimetic structures incorporating osf-1 or osteoinductive factors from Saos-2 cells indicates their potential for de novo bone formation that exploits cell-matrix interactions.

Intrauterine exposure to a maternal low protein diet reduces adult bone mass and alters growth plate morphology in rats.

Epidemiological studies suggest that poor growth during fetal life and infancy is associated with decreased bone mass in adulthood. However, theses observations have not, to date, been corroborated in animal models. To address this issue we evaluated the influence of maternal protein restriction on bone mass and growth plate morphology among the adult offspring, using a rat model. Maternal protein restriction resulted in a reduction in bone area and BMC, but not BMD, among the offspring in late adulthood. The widened epiphyseal growth plate in the protein-restricted offspring is compatible with the programming of cartilage and bone growth by maternal nutrition in early life.

Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification.

The ability to generate new bone for skeletal use is a major clinical need. Biomimetic scaffolds that interact and promote osteoblast differentiation and osteogenesis offer a promising approach to the generation of skeletal tissue to resolve this major health-care issue. In this study we examine the ability of surface-modified poly(lactic acid) (PLA) films and poly(lactic-co-/glycolic acid) (PLGA) (75:25) porous structures to promote human osteoprogenitor adhesion, spreading, growth, and differentiation. Cell spreading and adhesion were examined using Cell Tracker green fluorescence and confocal microscopy. Osteogenic differentiation was confirmed with alkaline phosphatase activity as well as immunocytochemistry for type I collagen, core binding factor-1 (Cbfa-1), and osteocalcin. Poor cell growth was observed on nonmodified PLA films and PLGA scaffolds. The polymers were then coupled with RGD peptides [using poly(L-lysine), or PLL] and physical adsorption as well as PLA films presenting adsorbed fibronectin (FN). Both modifications enhanced cell attachment and spreading. On PLA-FN and PLA-PLL-GRGDS films, the osteoblast response was dose dependent (20 pmol/L to 0.2 micromol/L FN and 30 nmol/L to 30 micromol/L PLL-GRGDS) and significant at concentrations as low as 2 nmol/L FN and 30 nmol/L PLL-GRGDS. With optimal concentrations of FN or RGD, adhesion and cell spreading were comparable to tissue culture plastic serum controls. In PLGA (75:25) biodegradable porous scaffolds, coated with FN, PLL-GRGDS, or fetal calf serum for 24 h in alpha MEM alone, prior to growth in dexamethasone and ascorbate-2-phosphate for 4-6 weeks, extensive osteoblast impregnation was observed by confocal and fluorescence microscopy. Cell viability in extended culture was maintained as analyzed by expression of Cell Tracker green and negligible ethidium homodimer-1 (a marker of cell necrosis) staining. Alkaline phosphatase activity, type I collagen, Cbfa-1, and osteocalcin expression were observed by immunocytochemistry. Mineralization of collagenous matrix took place after 4 weeks, which confirmed the expression of the mature osteogenic phenotype. These observations demonstrate successful adhesion and growth of human osteoprogenitors on protein- and peptide-coupled polymer films as well as migration, expansion, and differentiation on three-dimensional biodegradable PLGA scaffolds. The use of peptides/proteins and three-dimensional structures that provide positional and environmental information indicate the potential for biomimetic structures coupled with appropriate factors in the development of protocols for de novo bone formation.

Histological abnormalities of the calcaneum in congenital talipes equinovarus.

Six calcaneal fragments from patients aged 2, 3, 4, and 5 years with relapsed talipes, and two normal feet from a 40-week-old stillborn fetus were studied. All tissue was sectioned in the sagittal or coronal plane and stained using alcian blue and sirius red to distinguish cartilage and bone. Immunocytochemistry was performed to illustrate collagen types I and II. Within the clubfoot calcaneum, there were fewer chondrocytes and a diminished number of cartilage canals. Although a growth plate was present, the zones of differentiated chondrocytes were not apparent and the chondrocytes were smaller and flatter. The alcian blue staining within the spherical physis was paler than normal, suggesting that the amount of extracellular proteoglycans was reduced. Overall, the growth plate region of the talipes calcaneum resembled that of a permanent cartilage, like articular cartilage. Abnormalities were also seen in the ossification center. Cartilage spicules were rare, and developing bone frequently abutted directly onto the growth plate cartilage. The relative absence of a primary spongiosa suggested that the physis was virtually inactive and endochondral bone formation was retarded. These findings are consistent with the hypothesis that an intrinsic primary growth disorder causes the formation of a small hypoplastic bone and, subsequently, a smaller foot.