In vitro anti-cancer effects of the actin-binding natural compound rhizopodin.

Several natural compound interfere with microtubules or the actin cytoskeleton. Compounds interfering with the microtubules like Vinca-alkaloids or taxanes, are extensively used for cancer therapy. In contrast, knowledge about pharmacological properties of actin binding drugs is poor and drugs interfering with actin are far from clinical use. Rhizopodin is a natural compound that strongly affects the actin cytoskeleton at nanomolar concentrations. Initial work revealed interesting anti-bacterial and cytotoxic effects, but the cellular effects and pharmacological properties of rhizopodin have not been characterized. We hypothesized that rhizopodin might exert anti-cancer activity. Therefore, the aim of this study was to characterize the cellular and pharmacological effects of rhizopodin in cancer. Effects of rhizopodin demonstrated prominent effects on the actin cytoskeleton as shown in the actin-pyrene assay and by immunostaining of cancer cells. To investigate cellular effects of rhizopodin, we analyzed cell proliferation, cell death induction by propidium iodide exclusion and western blot, as well as migration by impedance measurement using the xCELLligence device in MDA-MB-231 breast cancer and T24 bladder cancer cell lines. Rhizopodin inhibited proliferation and induced cell death of MDA-MB-231 and T24 cells at nanomolar concentrations. PARP cleavage by rhizopodin suggests caspase-dependent cell death induction. Importantly, rhizopodin potently inhibited MDA-MB-231 and T24 cancer cell migration at subtoxic doses where no actin aggregation was observed, indicating a specific underlying signaling of rhizopodin. In summary, our study elucidates rhizopodin as actin-binding natural compound that exerts potent anti-cancer effects. Therefore, our work provides the basis for further in depth characterization of rhizopodin as an antitumoral agent.

Stereoselective total synthesis of axially chiral natural products via biaryl lactones.

Axially chiral natural products are rewarding synthetic targets, due to their wide distribution, diverse structures, and promising bioactivities. The "lactone concept" provides an efficient strategy for the regio- and stereoselective construction of even bulky biaryls. Key steps are the intramolecular coupling of the ester-prefixed molecular portions to give (mostly configurationally unstable) biaryl lactones and their stereoselective ring cleavage (usually by dynamic kinetic resolution), leading to the one or-optionally-the other atropisomeric product from the same lactone. Stereoisomeric byproducts can be recycled by recyclization back to the lactone. The broad applicability of the method is demonstrated in the total synthesis of selected representatives from five very different classes of natural biaryl products.

Inflammatory foreign-body reaction to an arthroscopic bioabsorbable meniscal arrow repair.

Various arthroscopic meniscal repair techniques have been developed in recent years to preserve meniscal function. We report the case of a patient with a failed arthroscopic meniscal repair demonstrating an inflammatory foreign-body reaction to bioabsorbable meniscal arrows.

Antiplasmodial activity of knipholone and related natural phenylanthraquinones.

phenylanthraquinone knipholone (1) and three of its natural derivatives as well as seven structurally related but simplified compounds have been examined for their antiplasmodial activity against asexual erythrocytic stages of two strains of Plasmodium falciparum in vitro (K1/chloroquine-resistant and NF 54/chloroquine-sensitive). All the phenylanthraquinones showed considerable activity with only little cytotoxicity, while their anthraquinone and phenyl moieties were completely inactive. Knipholone (1) and its natural derivatives can therefore be considered as a new group of potential antimalarials

The treatment of isolated articular cartilage lesions in the young individual.

The treatment of isolated articular cartilage defects is an evolving field in orthopaedic surgery today. We have summarized the basic science and clinical date on the treatment of isolated articular cartilage defects. Further long-term controlled studies are required in order to compare definitively the efficacy of treatments in this difficult clinical area. In future studies, inclusion/exclusion criteria must be detailed, and classification systems need to be standardized Comparative analysis can then be performed to assess the efficacy of various techniques.

Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair.

We have developed a novel, two-layered, collagen matrix seeded with chondrocytes for repair of articular cartilage. It consists of a dense collagen layer which is in contact with bone and a porous matrix to support the seeded chondrocytes. The matrices were implanted in rabbit femoral trochleas for up to 24 weeks. The control groups received either a matrix without cells or no implant. The best histological repair was seen with cell-seeded implants. The permeability and glycosaminoglycan content of both implant groups were nearly normal, but were significantly less in tissue from empty defects. The type-II collagen content of the seeded implants was normal. For unseeded implants it was 74.3% of the normal and for empty defects only 20%. The current treatments for articular injury often result in a fibrous repair which deteriorates with time. This bilayer implant allowed sustained hyaline-like repair of articular defects during the entire six-month period of observation.

A comparison of abrasion burr arthroplasty and subchondral drilling in the treatment of full-thickness cartilage lesions in the rabbit.

The purpose of this study was to observe the difference in healing of full-thickness articular cartilage defects treated with burr arthroplasty versus subchondral drilling. Cartilage was shaved off the medial femoral condyles of 39 rabbits without penetrating the subchondral plate. In left knees, two 2.0-mm holes were drilled into the condyle until bleeding was obtained. Right knees underwent a burr arthroplasty until punctate bleeding was observed. Animals were sacrificed at 6, 12, and 24 weeks postoperatively. Joint resurfacing and degenerative changes were evaluated grossly and histologically. Degenerative changes in the cartilage surface were observed with both treatments. Rabbits undergoing subchondral drilling had increased fibrocartilaginous healing with time, with a slight increase in degenerative changes. With burr arthroplasty, there was significant decrease in cartilaginous coverage of the exposed surface as well as progressive increase in degenerative changes. Although both techniques were suboptimal, histological evidence at 6 months suggests that subchondral drilling may result in a longer-lived repair than abrasion arthroplasty in the treatment of full-thickness lesions.

Repair of articular cartilage defects with collagen-chondrocyte allografts.

This study was designed to evaluate the potential use of a prototype collagen-chondrocyte allograft in the repair of full-thickness articular cartilage defects. Articular cartilage was harvested from young donor New Zealand White rabbits, enzymatically digested, cultured in monolayer, and passed into a three-dimensional porous type I collagen sponge (American Biomaterials). The composite grafts were incubated for 1 week. (Phase I) Twenty adult NZW rabbits underwent bilateral knee arthrotomies. Three-millimeter full-thickness articular cartilage defects were made in the trochlea of the distal femur. A 4-mm circular punch from the composite cell-seeded grafts was press-fit into the right knee defects. The left knee served as a control (collagen sponge alone or ungrafted defect). Animals were allowed free activity postoperatively and were killed in groups of five at 4, 8, 12, and 24 weeks postoperatively. Defect areas were harvested. Sections were cut at 5-microm thickness and stained with hematoxylin and eosin. The degree as well as quality of healing were assessed and scored with a grading system modified from Salter and O'Driscoll for cartilage repair. (The maximum score was 24 points.) Safranin-O staining as well as polarized light examination of representative sections was undertaken to assess the proteoglycan content and structural characteristics of the repair matrix. (Phase II) An additional 15 NZW rabbits underwent the above procedure but with the addition of fibroblast growth factor (FGF) (100 ng/ml) and insulin (5 microg/ml) to the growth medium of the composite grafts as stimulators of chondrocyte proliferation and proteoglycan synthesis. Control specimens in phase I and II (collagen sponge alone or ungrafted defects) healed with a primarily fibrous or fibrocartilagenous matrix. Defects grafted with cell-seeded collagen sponges demonstrated enhanced healing at all time points examined when compared to controls. There was a strong tendency toward a hyaline appearing matrix with increased Safranin-O staining and birefringence under polarized light more closely resembling the normal native cartilage. Mean histologic score for grafted defects was 18.4 (+/-3.1). Mean scores for collagen sponge alone and ungrafted defects in phase I were 12.7 (+/-4) and 12.7 (+/-3.1) (P<0.01). The addition of FGF and insulin to the growth medium (phase II) resulted in a significantly enhanced repair matrix when compared to the non-FGF-enhanced grafts, with a greater percentage of hyaline appearing tissue at all time points examined (4,8, and 12 weeks). Organization of the chondrocytes was improved at all time points examined as well. Mean histologic score for the FGF-grafted defects was 21.1 (+/-3.0). Mean scores for collagen sponge alone and ungrafted defects in phase II were 14.9 (+/-2.9) and 15.5 (+/-1.9) (p<0.01).

Use of the "blue dot" in femoral bone plug of bone-patellar tendon-bone graft in anterior cruciate ligament reconstruction.

The advancement of the femoral bone plug, with proper orientation through the femoral tunnel is often one of the most difficult maneuvers in the reconstruction of the anterior cruciate ligament using a bone-patellar tendon-bone graft. The authors describe a modification to the procedure featuring the use of methylene blue marker (to make a ¿blue dot¿) to highlight the site of drill holes as well as the anterior bone-tendon junction. The ¿blue dot¿ aids the localization of the drill hole with a probe and thus the advancement of the femoral plug through the femoral tunnel.

The effect of low-level Nd:YAG laser energy on adult articular cartilage in vitro.

Reports of laser energy applied to soft tissues in vitro and in vivo suggest both stimulation and inhibition of specific metabolic processes, depending on the type of laser, the energy density (ED) used, the mode of delivery, and type of tissue studied. An earlier in vitro study of Nd:YAG laser irradiation of articular cartilage indicated stimulation of both matrix and DNA synthesis for 6 days following laser exposure. In vivo reports on the ability of Nd:YAG laser energy to stimulate the healing of partial-thickness cartilage defects are conflicting. In the present study, a noncontact continuous-wave Nd:YAG laser beam of varying EDs was applied to full-thickness adult articular cartilage explants maintained in organ culture; the metabolic processes of chondrocyte DNA synthesis and matrix synthesis were followed over 2 weeks. For both canine and bovine cartilage, low-levels of laser energy (ED 51-127 J/cm2) stimulated matrix synthesis at 6-7 days following laser exposure, with a concomitant decrease in baseline DNA synthesis. By 12-14 days, however, these dose-dependent effects were no longer seen, with no significant differences from control noted for any of the laser energies studied. Histologic analysis of the cartilage explants following laser exposure showed no significant differences in cell number or morphology between sample and control groups; however, a decrease in matrix proteoglycan staining was seen in the highest laser energy group at all time points. These findings indicate that exposure to low-level noncontact Nd:YAG laser energy promotes a significant stimulation of cartilage matrix synthesis. However, a single exposure may not be sufficient to promote a sustained upregulation of cartilage metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

The intraoperative evaluation of the neurosensory function of the anterior cruciate ligament in humans using somatosensory evoked potentials.

Most of the investigation of the properties of the anterior cruciate ligament (ACL) has focused on its biomechanical functions. There has been increasing interest in the study of the possible neuroreceptor function of the ACL and its role in providing important proprioceptive feedback. Anatomic and histologic studies in humans have shown the presence of neuroreceptors within the anterior cruciate ligament. Indirect evidence exists that proprioception is diminished in the ACL-deficient knee. However, direct evidence in humans of the actual origin of the afferent impulses from within the ACL itself is lacking. Measurement of direct electrical afferent activity, occurring on stimulation of the ACL, should provide this evidence. Somatosensory evoked potentials (SEP) measure the electric potentials evoked in the cerebral cortex upon stimulation of a peripheral neuroreceptor. Carried by the posterior columns of the spinal cord, they reflect activity of the proprioceptive fibers. During arthroscopic procedures performed on nine patients, the normal ACL was stimulated by the use of electrodes applied to the femoral end, midsubstance, and tibial end, and cortical potentials thus evoked were recorded. In all cases, SEPs were recorded at the cerebral cortex upon stimulation of the ACL. The greatest potentials were reported upon stimulation of the midsubstance of the ligament. These findings provide direct evidence for, and strongly support the presence of, active proprioceptive receptors within the intact anterior cruciate ligament of the human knee.

Entrapment of an accessory superficial peroneal sensory nerve.

A 29 year old man had an accessory branch of the superficial peroneal nerve which entered the foot by rostro-caudally traversing the lateral malleolus laterally. The nerve was entrapped by a fascial band, resulting in pain over the lateral malleolus and dorsum of foot. Symptoms resolved when the nerve was surgically released.

The use of adhesives in chondrocyte transplantation surgery: in-vivo studies.

Two commercial adhesive preparations--fibrin glue and mussel adhesive protein (MAP)--were tested in-vivo for their ability to fix a chondrocyte allograft internally. While results for the fibrin, including additional testing for chondro inductive/conductive properties, were at best inconclusive, the results for MAP are highly promising.

The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation.

Using the knee joints of New Zealand White rabbits, a baseline study was made to determine the intrinsic capability of cartilage for healing defects that do not fracture the subchondral plate. A second experiment examined the effect of autologous chondrocytes grown in vitro on the healing rate of these defects. To determine whether any of the reconstituted cartilage resulted from the chondrocyte graft, a third experiment was conducted involving grafts with chondrocytes that had been labeled prior to grafting with a nuclear tracer. Results were evaluated using both qualitative and quantitative light microscopy. Macroscopic results from grafted specimens displayed a marked decrease in synovitis and other degenerative changes. In defects that had received transplants, a significant amount of cartilage was reconstituted (82%) compared to ungrafted controls (18%). Autoradiography on reconstituted cartilage showed that there were labeled cells incorporated into the repair matrix.

The problem of evaluating in situ pinning of slipped capital femoral epiphysis: an experimental model and a review of 63 consecutive cases.

Over a 3-year follow-up period, 63 hips (in 49 patients) that were pinned as treatment for slipped capital femoral epiphysis were examined and evaluated. A 36.8% incidence of unsuspected pin penetration was discovered. Four types of experimental models representing different degrees of severity of slipped capital femoral epiphysis were designed and manufactured in the bioengineering laboratory. In situ pinning was performed on each model. An extensive series of controlled test films on the models indicated the difficulty of accurately determining the true position of the pins with conventional roentgenographic views. Subsequent fluoroscopic analysis revealed a verifiable correlation between the limited visualization of conventional X-ray analysis following the pinning of a slipped capital femoral epiphysis and unrecognized pin penetration.

Platelets and microtubules. Effect of colchicine and D2O on platelet aggregation and release induced by calcium ionophore A23187.

We examined the role of microtubules in platelet aggregation and secretion (release reaction) induced by the calcium ionophore A23187 (0.8-5 muM). At these concentrations, platelet aggregation was preceded by a lag period of approximately 1 min. Colchicine (an agent that disrupts microtubule assembly-disassembly) was shown to bind to platelet microtubules by employing [(3)H]colchicine at a concentration that is specific for microtubules in other tissues (10 nM). Colchicine prolonged the lag period, inhibited the secondary wave of platelet aggregation, and inhibited the release reaction (release of [(14)C]serotonin). Platelets were next incubated with 20-60% D(2)O, an agent that stabilizes microtubules. D(2)O overcame colchicine-induced inhibition of the lag period, aggregation, and release. D(2)O alone enhanced platelet aggregation by 59+/-14% (SEM) and shortened the lag period by 43+/-10%. We conclude that functioning microtubules are required for platelet aggregation and release, and that microtubules of platelet preparations are functioning submaximally.