Tucci M

β class II tubulin predominates in normal and tumor breast tissues

BackgroundAntimitotic chemotherapeutic agents target tubulin, the major protein in mitotic spindles. Tubulin isotype composition is thought to be both diagnostic of tumor progression and a determinant of the cellular response to chemotherapy. This implies that there is a difference in isotype composition between normal and tumor tissues.MethodsTo determine whether such a difference occurs in breast tissues, total tubulin was fractionated from lysates of paired normal and tumor breast tissues, and the amounts of β-tubulin classes I + IV, II, and III were measured by competitive enzyme-linked immunosorbent assay (ELISA). Only primary tumor tissues, before chemotherapy, were examined. Her2/neu protein amplification occurs in about 30% of breast tumors and is considered a marker for poor prognosis. To gain insight into whether tubulin isotype levels might be correlated with prognosis, ELISAs were used to quantify Her2/neu protein levels in these tissues.Resultsβ-Tubulin isotype distributions in normal and tumor breast tissues were similar. The most abundant β-tubulin isotypes in these tissues were β-tubulin classes II and I + IV. Her2/neu levels in tumor tissues were 5–30-fold those in normal tissues, although there was no correlation between the Her2/neu biomarker and tubulin isotype levels.ConclusionThese results suggest that tubulin isotype levels, alone or in combination with Her2/neu protein levels, might not be diagnostic of tumorigenesis in breast cancer. However, the presence of a broad distribution of these tubulin isotypes (for example, 40–75% β-tubulin class II) in breast tissue, in conjunction with other factors, might still be relevant to disease progression and cellular response to antimitotic drugs.

Delivery of antifungal agents using bioactive and nonbioactive bone cements

Background: Management of fungal osteomyelitis is prolonged and frequently unsuccessful. Antifungal-impregnated cement is sometimes used as adjunctive therapy. Objective: To examine the release of antifungals from biodegradable and nonbiodegradable cement carriers. Methods: In vitro methods were used to assess antifungal drug release and antifungal activity of impregnated cements commonly used as adjunctive treatment of osteomyelitis. Cements included thermoplastic, nonbioactive polymers (polymethylmethacrylate [PMMA]) or bioactive agents (hydroxyapatite [HAP], β-tricalcium phosphate [β-TCP]) and were formed into spheres (beads). Results: Amphotericin B provided consistent supernatant concentrations (release), between 1.75 and 2.0 ug/mL, over 110 days from all bone cements. Flucytosine and fluconazole were observed for 33–42 days before becoming undetectable from a nonbioactive sphere and 18–22 days from a bioactive sphere. Serum concentrations for micafungin, terbinafine, and anidulafungin impregnated into PMMA rapidly became undetectable, regardless of the matrix used. Investigational β-TCP spheres prolonged release for fluconazole and micafungin, but had no effect on amphotericin B. Serum calcium concentrations decreased 60–80% in all HAP-impregnated drug sphere supernatants. Only amphotericin B–impregnated PMMA impacted supernatant calcium, decreasing concentrations by 50–60%. The antifungal-impregnated beads did not appear to be toxic to osteoblasts during 72 hours of exposure in tissue culture medium. Conclusions: Elution characteristics of most antifungals from bone cement spheres are probably not optima) for treatment of deep-seated fungal infections if a similar phenomenon of antifungal release manifests in vivo. Ceramic nonabsorbable impregnated devices must be removed after their lifespan expires and may necessitate another surgical procedure that can increase surgical risk and cost. Bioactive osteoconductive materials may provide a surgical alternative to nonabsorbable matrices. However, there have been no controlled trials demonstrating improved therapeutic outcomes with local therapy and assessing whether biodegradable materials act as a new focus for infection.

Characterization of a Femoral Segmental Nonunion Model in Laboratory Rats: Report of a Novel Surgical Technique

The literature is lacking conclusive results regarding the exact mechanism of maximizing the fracture healing stages with minimal traumatic side effects. This observation mandates the development of a novel surgical procedure using small animals as a model to study fracture healing in the presence of osteoinductive agents. Previously, stabilization of osteotomies in small animal models has mainly been accomplished using Kirschner wires, but the rats tremendous ability to heal an osteotomy stabilized by this method has masked the effects of osteoinductive agents. Thus, this study proposes using a modified 20-hole, 1.5-mm stainless-steel plate to stabilize a 5-mm segmental defect. Thirty of 32 adult male rats were fully weight-bearing within 2 days and were followed over a 15-week period. Two animals showed evidence of fixation failure due to technical error, and the animals were humanely sacrificed. At the end of the study, the fractures were stable with significantly less bone formation evident when compared to controls (p <. 001). Therefore, this technique can effectively be used to evaluate compounds that will enhance bone formation and allows for stable fixation of the control with minimal callus formation or bony ingrowth. The goal of this article is to allow other investigators to reproduce this technique as well as outline the advantages and disadvantages of this novel plating technique versus the former Kirschner wire technique for the study of osteoinductive agents using small animals as a model.

Lipopolysaccharide-stimulated nitric oxide production and inhibition of cell proliferation is antagonized by ethanol in a clonal macrophage cell line

Both chronic and acute ethanol exposure have been shown to be cytotoxic and also to disrupt normal cell function or responses in a variety of cell types. Macrophage function has specifically been shown to be disrupted by chronic ethanol exposure by mechanisms that have not been elucidated. It is known that exposure of macrophages to lipopolysaccharide (LPS) from gram-negative bacteria will decrease the number of cells. Since increased exposure to endotoxin is often associated with chronic alcoholism, this may be one mechanism to account for loss of macrophages in alcoholic patients. The loss of macrophages, as a consequence of endotoxin treatment, appears to be linked to cell activation and, in particular, LPS-stimulated synthesis of nitric oxide which has been suggested to cause an increase in apoptosis. Ethanol also increases apoptosis in some cell types but, in general, ethanol inhibits activation of macrophages. Thus, the overall effect on cell numbers and cell proliferation elicited by treating macrophages concomitantly with ethanol and LPS depends on the balance between inhibiting LPS-mediated activation and the actions of ethanol. The interaction between ethanol and LPS was investigated in a macrophage cell line (RAW 264.7 cells) by measuring nitric oxide production and cell proliferation. A 24-h exposure to ethanol (100 mM) decreased [3H]-thymidine incorporation significantly. LPS treatment elicited a concentration-dependent decrease in [3H]-thymidine incorporation at LPS concentrations of 0.1 ng/ml to 1000 ng/ml and stimulated nitric oxide production at concentrations above 1 ng/ml. LPS-stimulated nitric oxide production was inhibited by ethanol (20 to 100 mM) and the nitric oxide synthesis inhibitor, N(G)Nitro-L-arginine methyl L-NAME) ester (100 and 500 microM). However, LPS-inhibited [3H]-thymidine incorporation was not be totally reversed by ethanol- or L-NAME-treatment. A direct correlation between nitric oxide production and inhibition of cell proliferation could not be demonstrated. However, it appears that ethanol and LPS do affect some common mechanism(s) in this cell line.

Comparison of Two Different Fixation Techniques for a Segmental Defect in a Rat Femur Model

Studies have attempted to identify the osteogenic effects of bone morphogenetic proteins using a rat femur model, which commonly involves the creation of a critical size defect followed by internal fixation of the femur. Among the most familiar fixation methods are either plating or intramedullary placement of a Kirschner wire (K-wire). There are advantages and disadvantages to each method; however, this study attempts to identify the best method by exploring the histological effects of each technique. The experiment involved two groups with no added treatment: Group P (plate fixation method) and Group K (K-wire fixation method). The animals were allowed a 4-week interval for the femurs to heal, and proximal, distal, and two midshaft cuts were examined under high-power microscopy after the fixation apparatus was removed. Group K exhibited a peculiar fibrotic healing pattern that followed the shaft of the then vacated K-wire and there was minimal new viable bone formation. Group P, however, exhibited a more natural ingrowth of newly formed bone that began at the proximal and distal cuts and proceeded centrally into the core of the defect. Due to the fibrotic tissue in Group K, this study shows that the model is insufficient due to the micromotion created and thus supports plating of critical defects as the fixation method of choice due to the creation of a stable healing environment.

Dynamic anterior cervical plating for multi-level spondylosis: Does it help?

Study design: Randomized controlled trial. Objective: To compare fusion rates, time to fusion, complication rates and subsidence between 1) a static, 2) a dynamic angulation, and 3) a dynamic translation plate in anterior cervical discectomy and fusion for symptomatic degenerative cervical disease. Methods: Thirty-six patients with two level, symptomatic cervical degenerative changes requiring surgery were randomized in a blinded fashion to receive a statically locked plate, Cervical Spine Locking Plate (CSLP) (Synthes, Paoli, PN, USA), an Atlantis Vision® Anterior Cervical Plate System (Medtronic, Memphis, TN, USA) which allows angular dynamization, or a Premier® Anterior Cervical Plate System (Medtronic) which allows translational dynamization. Structured data collection and measurement protocols were used. Intervertebral composite allograft cages were used in all groups. Identical external immobilization and antiinflammatory medication protocols were followed. X-rays were obtained at preset time points postoperatively. Assessment of the primary outcomes was blinded. Rate of and time to fusion, graft/instrumentation complications, subsidence, and reoperation for adjacent level disease were measured. Paired t-test and three-way Analysis of Variance test (ANOVA) were used to assess statistical differences between groups. Results: The three groups were similar demographically. Fusion rates in the CSLP, Atlantis and Premier plate groups were 100%, 91%, and 92% respectively. Mean time to fusion was 6.1, 8.3 and 6.3 months respectively but differences were not statistically significant. Mean subsidence in the groups was 1.9, 1.6, and 2.6 mm respectively. Subsidence was found even for the static (CSLP) plate, but no statistically significant differences were found. Conclusions: We found no clinical advantage of dynamic plates over static plates with regards to fusion rates, time to fusion, subsidence, complications, or adjacent-level surgery. Static plating allows for subsidence at similar levels to dynamic plating. Methods evaluation and class of evidence (CoE) Methodological principle: Study design: RCT • Cohort Case control Case series Concealed allocation (RCT) Intent to treat (RCT) • Blinded/independent evaluation of primary outcome • Complete follow-up of ≥85%* • Adequate sample size Control for confounding† • Evidence class: II *Reliable data are data such as mortality or reoperation. †Authors must provide a description of robust baseline characteristics, and control for those that are unequally distributed between treatment groups. The definiton of the different classes of evidence is available on page 83.

Animal Models of Corticosteroid- Associated Bone Diseases

Osteonecrosis is a disease that is characterized by lesions of dead bone, most frequently detected in the subchondral bone of the convex side of the major diarthrodial joints (hips, knees, shoulders, and talus). High-dose corticosteroid therapy is one major risk factor for osteonecrosis. The pathological appearance of osteonecrosis of the femoral head, however, is fairly consistent regardless of the etiology. Depending on the stage of the disease, this includes evidence of bone marrow edema, lipocyte and bone cell necrosis, fi brosis, creeping substitution (new bone lying on dead bone), and the presence of osteoporosis [ 1 ]. Hemorrhage within the bone marrow compartment has also been observed in patient specimens from corticosteroid-associated osteonecrosis [ 2 ]. Previous investigators have suggested that corticosteroid- associated osteonecrosis has more rapid progression than other etiologies [ 1 , 3 ]. The mechanisms involved in the development of osteonecrosis [ON] are diffi cult to explore from clinical samples as they provide a single snapshot at one time point subsequent to the onset of the disease, usually months after the onset of the disease when the symptoms fi rst appear. While some investigators have used corticosteroid- associated animal models to evaluate different potential treatments [ 4 ‐ 6 ], it is not clear whether the results can be extrapolated to the human condition. A better understanding of the pathophysiological mechanisms is needed. An animal model of corticosteroid-associated osteonecrosis would allow us to study the pathogenesis, diagnosis, and treatment intervention of this specifi c etiology. There have been a number of attempts to establish a corticosteroidassociated animal model ‐ primarily focusing on giving different species of animals large doses of corticosteroids such as dexamethasone, prednisolone, and prednisone for various periods of time. Yet, no progressive ON is established with evidence of creeping substitution and subchondral collapse and degenerative joint disease with corticosteroid administration alone. These factors that may confound the development of corticosteroid-associated ON animal models include differences in weight bearing (quadruped vs. bipedal), age, length of treatment, comorbidities, and differences in metabolic pathways. In spite of these differences, these animal models can be used to evaluate the effect of exogenous corticosteroid administration on bone, its vasculature, and other organs. Selected studies used to evaluate the effect of corticosteroid treatment on bone are listed in Table 64.1 .

The HL-60 Cells Response to Various Particle Sizes of Tetracalcium Phosphate Ceramic Delivery System

The specific objective of this investigation is to study the effect of tetracalcium phosphate (TeCP) delivery system fabricated from three different particle sizes (<38, 45 and 75 um) on the viability of HL-60 cell line in culture. Each TeCP reservoir was fabricated using standard protocols. The sintered microcrystal material were cold pressed at 5000 kg compression load, sterilized and loaded into wells pre-plated with 106 HL-60 cells. At the end of 24, 48, and 72 hours, the supernatants (cell suspension) from the wells were collected for cell count (a hemacytometer method) and lactate dehydrogenase (LDH) assays. The cells were evaluated for structural cellular damage by using conventional histopathological protocols (H&E stain). Results obtained from this investigation suggest the followings: (i) particle sizes are key elements in developing TeCP delivery system, (ii) the use of <38 um particle sizes would be favorable for long duration of delivering biologicals from the TeCP reservoirs due to the viability of HL-60 cells, (iii) LDH activities were directly proportional the particle sizes of the ceramic capsules compared to the control and (iv) there were less cellular structural changes in devices fabricated from <38 um particle sizes and the cells became more reactive with an increase in particle sizes.

A new model for repair of the Achilles tendon in the rat.

ABSTRACT The Sprague-Dawley rat is an excellent model for studies of Achilles tendon repair. Most researchers use a modification of the Kessler technique for suture repair of the Achilles tendon in rats. While this technique provides adequate strength, early mobilization is not recommended. Prior to healing, the load will be borne completely by the suture repair, subjecting it to rupture. To prevent this complication, investigators employing the Kessler repair often immobilize the operative extremity with a cast or splint. This has also been shown to be detrimental to the peak load borne by the tendons prior to rupture. A double-loop locking technique of suture repair for rat Achilles tendons is favored over the modified Kessler technique. As force is applied across the repair, the suture pulls on the tendon, sharing the load. This allows for early mobilization of repaired tendons, with minimal risk of rupture. Additionally, no immobilization is required for the operative extremity. One hundred repairs have been performed using this double-loop locking technique. All animals have been able to mobilize with minimal limp immediately after recovering from anesthesia, and there have been no ruptures. No other complications have occurred (hematoma, seroma, infection, dehiscence). This technique of tendon repair is ideal for use in studies of tendon repair in the rat, since it is easy to perform and eliminates the need for immobilization of the operative leg.

Analysis Of Tissues Collected During Primary And Revision Hip Arthroplasty

that when foreign introduced into a host a sequence of biologicdl owing the marerial to become thin the tissues. The reactive form between the implant and bone an lead to late aseptic loosening. This type of loosening is implicated to be the most a m o n reason for implant failure. It is beIieved that more mature aseptic loosening involves abnormaI and even normal mechanical suesses can Iead to the production of m pic wear particles. Submicron wear p can either be trapped within the joint or to adjacent tissues where they can init de of inflammatory reactions that allow for the recruitment of M a , lymphs and with time a fibrous ,“doma like tissue will develop around the implant. The cell types found at the interface are capable of secreting bone-resorbing cytokines. Ln addition to the osteouophic factors (cytokines), biologicd oxidation of tissues has also been reco,onized as a primary event in the pad.lo,oenesis of many diseases. Reactive ies of O._ and hydroxyl radicals (OH? ar rmed within norma1 biologica1 tissues and propagate along the ’ phospholipid membranes which can cause dative damage of tissues. n e goal ‘of this ine the levels of cytokines and e herface tiSsues reuieved ,