Search

Total loss of talus due to trauma or avascular necrosis, for example, still remains to be a major challenge in foot and ankle surgery with severely limited treatment options. Implantation of a custom made total talar prosthesis has shown promising results so far. Most important factors for long time success are degree of congruence of articular surfaces and ligamentous stability of the ankle. Therefore, our aim was to develop an optimized custom made prosthesis for total talus replacement providing a high level of primary stability. A custom made hemiprosthesis was developed using computed tomography and magnetic resonance imaging data of the affected and contralateral talus considering the principles and technology for the development of the S.T.A.R. prosthesis (Stryker). Additionally, four eyelets for fixation of artificial ligaments were added at the correspondent footprints of the most important ligaments. Two modifications can be provided according to the clinical requirements: A tri-articular hemiprosthesis or a bi-articular hemiprosthesis combined with the tibial component of the S.T.A.R. total ankle replacement system. A feasibility study was performed using a fresh frozen human cadaver. Maximum range of motion of the ankle was measured and ligamentous stability was evaluated by use of standard X-rays after application of varus, valgus or sagittal stress with 150 N. Correct implantation of the prosthesis was technically possible via an anterior approach to the ankle and using standard instruments. Malleolar osteotomies were not required. Maximum ankle dorsiflexion and plantarflexion were measured as 22-0-28 degrees. Maximum anterior displacement of the talus was 6 mm, maximum varus tilt 3 degrees and maximum valgus tilt 2 degrees. Application of an internally braced prosthesis for total talus replacement in humans is technically feasible and might be a reasonable procedure in carefully selected cases with no better alternatives left., Competing Interests: Conflict-of-interest statement: The authors report no relevant conflicts of interest. Kevin Soldan and Steffen Peyerl are employees of Stryker (Selzach, Switzerland). Markus Regauer and Hans Polzer are paid consultants of Arthrex (Naples, FL, United States).

Introduction: Impaired ankle dorsiflexion (ADF) is known to increase forefoot pressure, which is associated to various pathologies affecting the foot and ankle. M. gastrocnemius tightness (MGT) is its most common cause. Up to date we are missing a standardized examination procedure, norm values, and a valid decision pathway to diagnose impaired ADF and MGT. The aim of this study was to define norm values for ADF using a standardized examination procedure. These were used to define a decision pathway to diagnose impaired ADF and MGT., Materials and Methods: 64 young, asymptomatic subjects were examined. Based on a standardized examination procedure, bilateral ADF, both with the knee extended and flexed, non-weight bearing and weight bearing, was assessed by three investigators. Inter-rater test reliability and norm values for ADF were calculated. Side differences were analyzed. ADF differences between the knee extended and flexed were calculated., Results: The standardized examination procedure revealed high ICC values (0.876-0.915). ADF values with the knee extended for the left/right limb were 22.7° ± 5.9° [95 % CI 21.2°-24.3°]/23.4° ± 6.5° [95 % CI 21.7°-25.1°] non-weight bearing and 33.3° ± 5.5° [95 % CI 31.9°-34.7°]/33.6° ± 5.6° [95 % CI 32.1°-35.0°] weight bearing. Physiological side differences with the knee extended were <6° (95 % CI). Knee flexion resulted in an approximate ADF increase of 10°., Conclusions: Based on an extensive systematic approach, physiological values for ADF were assessed in a large asymptomatic population. This allowed the definition of a decision pathway to diagnose impaired ADF and MGT. Patients presenting with pathologies associated with impaired ADF should be examined according to the herein presented examination protocol. This systematic approach provides a consistent definition of impaired ADF and MGT, which is the prerequisite to study the effectiveness of treatment strategies for MGT.

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described to assess MGT. Still, a standardized examination procedure is missing. Literature argues for weightbearing examination but the degree of knee flexion needed to eliminate the restraining effect of the M. gastrocnemius on ankle dorsiflexion (ADF) is unknown. This manuscript investigates the effect of knee flexion on ankle dorsiflexion and provides a detailed description of a standardized examination protocol. Examination on 20 healthy individuals revealed, that 20° of knee flexion is sufficient to fully eliminate the influence of the M. gastrocnemius on ADF. This builds the prerequisite for a standardized examination for MGT. Non-weightbearing and weightbearing examination of ADF has to be conducted with the knee fully extended and at least 20° flexed. Two investigators should conduct non-weightbearing testing with the subject in supine position. In order to obtain reliable results, the axis of the fibula should be marked. One examiner can conduct weightbearing examination with the subject in lunge stance. Isolated MGT is present if ADF is impaired with the knee fully extended and knee flexion results in a significant ADF increase. The herein presented standardized examination is the prerequisite for future studies aiming at establishing norm values.

In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor.

Background: An anatomical reconstruction of the ankle congruity is the important prerequisite in the operative treatment of acute ankle fractures. Despite anatomic restoration patients regularly suffer from residual symptoms after these fractures. There is growing evidence, that a poor outcome is related to the concomitant traumatic intra-articular pathology. By supplementary ankle arthroscopy anatomic reduction can be confirmed and associated intra-articular injuries can be treated. Nevertheless, the vast majority of complex ankle fractures are managed by open reduction and internal fixation (ORIF) only. Up to now, the effectiveness of arthroscopically assisted fracture treatment (AORIF) has not been conclusively determined. Therefore, a prospective randomised study is needed to sufficiently evaluate the effect of AORIF compared to ORIF in complex ankle fractures., Methods/design: We perform a randomised controlled trial at Munich University Clinic enrolling patients (18-65 years) with an acute ankle fracture (AO 44 A2, A3, B2, B3, C1 - C3 according to AO classification system). Patients meeting the inclusion criteria are randomised to either intervention group (AORIF, n = 37) or comparison group (ORIF, n = 37). Exclusion criteria are fractures classified as AO type 44 A1 or B1, pilon or plafond-variant injury or open fractures. Primary outcome is the AOFAS Score (American Orthopaedic Foot and Ankle Society). Secondary outcome parameter are JSSF Score (Japanese Society of Surgery of the Foot), Olerud and Molander Score, Karlsson Score, Tegner Activity Scale, SF-12, radiographic analysis, arthroscopic findings of intra-articular lesions, functional assessments, time to return to work/sports and complications. This study protocol is accordant to the SPIRIT 2013 recommendation. Statistical analysis will be performed using SPSS 22.0 (IBM)., Discussion: The subjective and functional outcome of complex ankle fractures is regularly unsatisfying. As these injuries are very common it is essential to improve the postoperative results. Potentially, arthroscopically assisted fracture treatment can significantly improve the outcome by addressing the intra-articular pathologies. Given the absolute lack of studies comparing AORIF to ORIF in complex ankle fractures, this randomised controlled trail is urgently needed to evaluate the effectiveness of additional arthroscopy., Trial Registration: ClinicalTrials.gov reference: NCT02449096 (Trial registration date: April 7th, 2015).

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase constitutively expressed by acute myeloid leukaemia (AML) blasts. In addition, 25% of AML patients harbour a FLT3-ITD mutation, associated with inferior outcome due to increased relapse rate. Relapse might be propagated by interactions between AML blasts and the bone marrow microenvironment. Besides cellular elements of the microenvironment (e.g. mesenchymal stromal cells), bone marrow hypoxia has emerged as an additional crucial component. Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology. Here we show that 25% of AML patients down-regulate FLT3 expression on blasts in response to in vitro hypoxia (1% O2), which was independent of its mutational state. While virtually no AML cell lines regulate FLT3 in response to hypoxia, the down-regulation could be observed in Ba/F3 cells stably transfected with different FLT3 mutants. Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia. Also, PI-3K inhibition could partially abrogate down-regulation of FLT3. Hypoxia-mediated down-regulation of FLT3 conferred resistance against cytarabine in vitro. In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.

The acute rupture of the Achilles tendon is a protracted injury. Surgery is only the beginning of a long rehabilitation period. Therefore, the rehabilitation protocol is an integral aspect to restore the pre-injury activity level. Despite several trials available comparing different treatment regimes, there is still no consensus regarding the optimal protocol. Consequently, the aim of our study was to systematically search the evidence available and define a precise rehabilitation programme after operative repair of acute Achilles tendon rupture based on the trials with the highest level of evidence. We performed a systematic literature search in Medline, Embase and Cochrane library. We identified twelve randomized controlled trials comparing different treatment regimes after operative repair of the Achilles tendon. Five trials compared full to non weight bearing, all applying immobilization in equinus. Immediate full weight bearing led to significant higher patient satisfaction, earlier ambulation and return to pre-injury activity. Four trials compared early ankle mobilization to immobilization. All trials found mobilization to be superior as it shortens time to return to work and sports significantly. Three trials compared the combination of full weight bearing and early ankle mobilization to immobilization. This combination was most beneficial. Patients showed significantly higher satisfaction, less use of rehabilitation resources, earlier return to pre-injury activities and further demonstrated significantly increased calf muscle strength, reduced atrophy and tendon elongation. No study found an increased rerupture rate for the more progressive treatment. In conclusion, the rehabilitation protocol after Achilles tendon repair should allow immediate full weight bearing. After the second postoperative week controlled ankle mobilization by free plantar flexion and limited dorsiflexion at 0° should be applied., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Musculus gastrocnemius tightness (MGT) can be diagnosed by comparing ankle dorsiflexion (ADF) with the knee extended and flexed. Although various measurement techniques exist, the degree of knee flexion needed to eliminate the effect of the gastrocnemius on ADF is still unknown. The aim of this study was to identify the minimal degree of knee flexion required to eliminate the restricting effect of the musculus gastrocnemius on ADF., Methods: Bilateral ADF of 20 asymptomatic volunteers aged 18-40 years (50% female) was assessed prospectively at six different degrees of knee flexion (0°, 20°, 30°, 45°, 60°, 75°, Lunge). Tests were performed following a standardized protocol, non weightbearing and weightbearing, by two observers. Statistics comprised of descriptive statistics, t-tests, repeated measurement ANOVA and ICC., Results: 20 individuals with a mean age of 27 ± 4 years were tested. No significant side to side differences were observed. The average ADF [95% confidence interval] for non weightbearing was 4° [1°-8°] with the knee extended and 20° [16°-24°] for the knee 75° flexed. Mean weightbearing ADF was 25° [22°-28°] for the knee extended and 39° [36°-42°] for the knee 75° flexed. The mean differences between 20° knee flexion and full extension were 15° [12°-18°] non weightbearing and 13° [11°-16°] weightbearing. Significant differences of ADF were only found between full extension and 20° of knee flexion. Further knee flexion did not increase ADF., Conclusion: Knee flexion of 20° fully eliminates the ADF restraining effect of the gastrocnemius. This knowledge is essential to design a standardized clinical examination assessing MGT.

Osteoid osteomas are typically located in the femur and tibia and are mostly easy to diagnose based on patient age, the clinical signs and plain radiographs. In contrast, the diagnosis of osteoid osteomas of the foot is often delayed because of the atypical presentation. We report the case of a 24-year-old patient with persisting pain in the ankle joint over 8 years due to an osteoid osteoma of the talus neck.

Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or inhibits osteogenic differentiation in a time dependent manner indicates an age related alteration in signal transduction of hMSC and requires further investigation.

About 30% of patients with acute myeloid leukemia (AML) harbour mutations of the receptor tyrosine kinase FLT3, mostly internal tandem duplications (ITD) and point mutations of the second tyrosine kinase domain (TKD). It was the aim of this study to comprehensively analyze clinical and functional properties of various FLT3 mutants. In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with a worse relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a strong activating phenotype in Ba/F3 cells. In contrast, FLT3-TKD mutations and other point mutations--including two novel mutations--showed a weaker but clear gain-of-function phenotype with gradual increase in proliferation and protection from apoptosis. The pro-proliferative capacity of the investigated FLT3 mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD positive cell lines, in contrast to FLT3-non-ITD mutants, which displayed an enhanced signal of AKT and MAPK activation. Gene expression analysis revealed distinct difference between FLT3-ITD and FLT3-TKD for STAT5 target gene expression as well as deregulation of SOCS2, ENPP2, PRUNE2 and ART3. FLT3-ITD and FLT3 point mutations show a gain-of-function phenotype with distinct signalling properties in vitro. Although poor prognosis in AML is only associated with FLT3-ITD, all activating FLT3 mutations can contribute to leukemogenesis and are thus potential targets for therapeutic interventions.

Hallux rigidus describes the osteoarthritis of the first metatarsophalangeal joint. It was first mentioned in 1887. Since then a multitude of terms have been introduced referring to the same disease. The main complaints are pain especially during movement and a limited range of motion. Radiographically the typical signs of osteoarthritis can be observed starting at the dorsal portion of the joint. Numerous classifications make the comparison of the different studies difficult. If non-operative treatment fails to resolve the symptoms operative treatment is indicated. The most studied procedure with reproducible results is the arthrodesis. Nevertheless, many patients refuse this treatment option, favouring a procedure preserving motion. Different motion preserving and joint sacrificing operations such as arthroplasty are available. In this review we focus on motion and joint preserving procedures. Numerous joint preserving osteotomies have been described. Most of them try to relocate the viable plantar cartilage more dorsally, to decompress the joint and to increase dorsiflexion of the first metatarsal bone. Multiple studies are available investigating these procedures. Most of them suffer from low quality, short follow up and small patient numbers. Consequently the grade of recommendation is low. Nonetheless, joint preserving procedures are appealing because if they fail to relief the symptoms an arthrodesis or arthroplasty can still be performed thereafter.

Scaffolds seeded with multipotent precursor cells were hypothesized to heal critically sized bone defects. However, the success of this concept was limited by low cell survival after transplantation due to a lack of nutrients and oxygen. In vivo prevascularization of scaffolds before cell seeding may improve cell survival, yet the best seeding technique and time point of cell application remain elusive. Thus, the aim of this study was to compare different strategies. Demineralized bone matrix scaffolds were implanted around the saphenous arteriovenous (AV) bundle in nude mice. In vivo seeding was performed 0, 5, or 21 days after implantation using enhanced green fluorescent protein (eGFP)-expressing mesenchymal stem cells (MSCs). Cells were applied either by injection or the repetitive dripping technique. In vitro seeded and subcutaneously implanted scaffolds served as controls. Fourteen days after cell application, the fluorescence intensity of transplanted cells and the extent of newly formed vessels were quantified. We found that the AV flow through model as well as cell application increased vessel formation. In vitro seeding resulted in significantly higher cell numbers than in vivo seeding. With increasing time of prevascularization, the number of cells declined dramatically. In vivo seeding by cell injection was superior to the repetitive dripping protocol. On subcutaneously implanted scaffolds, significantly, more cells were found than on axially perfused scaffolds. We conclude that in vitro seeding is more efficient compared to the two novel in vivo seeding techniques of prevascularized scaffolds. With increasing time of prevascularization, the seeding efficiency for the in vivo methods further decreases, presumably due to the ingrowth of connective tissue. Even though, the presence of MSCs and the longer period of prevascularization enhances vessel formation, this conceivable advantage is limited supposedly by the inferior seeding efficiency.

Osteoporotic fractures show reduced callus formation and delayed bone healing. Cellular sources of fracture healing are mesenchymal stem cells (MSC) that differentiate into osteoblasts by stimulation with osteoinductive cytokines, such as BMP-2. We hypothesized that impaired signal transduction and reduced osteogenic differentiation capacity in response to BMP-2 may underlie the delayed fracture healing. Therefore, MSC were isolated from femoral heads of healthy and osteoporotic patients. Grouping was carried out by bone mineral densitometry in an age-matched manner. MSC were stimulated with BMP-2. Signal transduction was assessed by western blotting of pSMAD1/5/8 and pERK1/2 as well as by quantitative RT-PCR of Runx-2, Dlx5, and Osteocalcin. Osteogenic differentiation was assessed by quantifying Alizarin Red staining. Osteoporotic MSC featured an accurate phosphorylation pattern of SMAD1/5/8 but a significantly reduced activation of ERK1/2 by BMP-2 stimulation. Furthermore, osteoporotic MSC showed significantly reduced basal expression levels of Runx-2 and Dlx5. However, Runx-2, Dlx5, and Osteocalcin expression showed adequate up-regulation due to BMP-2 stimulation. The global osteogenic differentiation in standard osteogenic differentiation media was reduced in osteoporotic MSC. Nevertheless, osteoporotic MSC were shown to feature an adequate induction of osteogenic differentiation due to BMP-2 stimulation. Taken together, we here demonstrate osteoporosis associated alterations in BMP-2 signaling but sustained specific osteogenic differentiation capacity in response to BMP-2. Therefore, BMP-2 may represent a promising therapeutic agent for the treatment of fractures in osteoporotic patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The t(8;21) and inv(16)/t(16;16) rearrangements affecting the core-binding factors RUNX1 and CBFB, respectively, are found in 15% to 20% of adult de novo acute myeloid leukemia (AML) cases and are associated with a favorable prognosis. Since the expression of the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1 alone is not sufficient to cause leukemia, we performed exome sequencing of an AML sample with an inv(16) to identify mutations, which may collaborate with the CBFB/MYH11 fusion during leukemogenesis. We discovered an N676K mutation in the adenosine triphosphate (ATP)-binding domain (tyrosine kinase domain 1 [TKD1]) of the fms-related tyrosine kinase 3 (FLT3) gene. In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). The FLT3-N676K mutant alone leads to factor-independent growth in Ba/F3 cells and, together with a concurrent FLT3-ITD (internal tandem duplication), confers resistance to the FLT3 protein tyrosine kinase inhibitors (PTKIs) PKC412 and AC220. Gene expression analysis of AML patients with CBFB/MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias.

In addition to overexpression and the occurrence of activating mutations, receptors can be aberrantly activated by impaired downregulation. In this study, we show that an oncogenic mutant of the ubiquitin ligase casitas B-lineage lymphoma (CBL; CBLΔexon8), which is found in acute myeloid leukemia patients, predominantly cooperates with receptor tyrosine kinase (RTK) class III receptors (PDGFRA, PDGFRB, KIT, and FLT3), but not with non-class III RTKs or cytokine receptors, to induce IL-3-independent growth of Ba/F3 cells. In cells coexpressing RTK class III/CBLΔexon8, receptor internalization was delayed, and cells were protected from apoptosis after cytokine withdrawal. Ligand-stimulated Ba/F3 cells and acute myeloid leukemia cell lines coexpressing the CBL deletion mutant and FLT3 showed enhanced AKT phosphorylation. Combined pharmacologic inhibition of the PI3K/AKT pathway and FLT3 had an additive effect on cell proliferation. The transforming potential of the CBL mutant was completely abolished by the mutation of the CBL PTB domain and was decreased by the mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutively active AKT1 mutant (E17K) recapitulated the phenotype induced by the CBL deletion mutant in Ba/F3 cells. This study reveals FLT3-CBL interaction sites and the AKT pathway as critical mediators of transformation by oncogenic CBL mutants., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Ankle sprains often result in ankle instability, which is most likely caused by damage to passive structures and neuromuscular impairment. Whole body vibration (WBV) is a neuromuscular training method improving those impaired neurologic parameters. The aim of this study is to compare the current gold standard functional treatment to functional treatment plus WBV in patients with acute unilateral unstable inversion ankle sprains., Methods/design: 60 patients, aged 18-40 years, presenting with an isolated, unilateral, acute unstable inversion ankle sprain will be included in this bicentric, biphasic, randomized controlled trial. Samples will be randomized by envelope drawing. All patients will be allowed early mobilization and pain-dependent weight bearing, limited functional immobilization by orthosis, PRICE, NSARDs as well as home and supervised physiotherapy. Supervised physical therapy will take place twice a week, for 30 minutes for a period of 6 weeks, following a standardized intervention protocol. During supervised physical therapy, the intervention group will perform exercises similar to those of the control group, on a side-alternating sinusoidal vibration platform. Two time-dependent primary outcome parameters will be assessed: short-term outcome after six weeks will be postural control quantified by the sway index; mid-term outcome after one year will be assessed by subjective instability, defined by the presence of giving-way attacks. Secondary outcome parameters include: return to pre-injury level of activities, residual pain, recurrence, objective instability, energy/coordination, Foot and Ankle Disability Index and EQ 5D., Discussion: This is the first trial investigating the effects of WBV in patients with acute soft tissue injury. Inversion ankle sprains often result in ankle instability, which is most likely due to damage of neurological structures. Due to its unique, frequency dependent, influence on various neuromuscular parameters, WBV is a promising treatment method for patients with acute unstable inversion ankle sprains., Trial Registration: NCT01702597.

Activating mutations of the class III receptor tyrosine kinase FLT3 are the most frequent molecular aberration in acute myeloid leukemia (AML). Mutant FLT3 accelerates proliferation, suppresses apoptosis, and correlates with poor prognosis. Therefore, it is a promising therapeutic target. Here, we show that RNA interference against FLT3 with an internal tandem duplication (FLT3-ITD) potentiates the efficacy of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) against AML cells expressing FLT3-ITD. Similar to RNA interference, tyrosine kinase inhibitors (TKI; AC220/cpd.102/PKC412) in combination with LBH589 exhibit superior activity against AML cells. Median dose-effect analyses of drug-induced apoptosis rates of AML cells (MV4-11 and MOLM-13) revealed combination index (CI) values indicating strong synergism. AC220, the most potent and FLT3-specific TKI, shows highest synergism with LBH589 in the low nanomolar range. A 4-hour exposure to LBH589 + AC220 already generates more than 50% apoptosis after 24 hours. Different cell lines lacking FLT3-ITD as well as normal peripheral blood mononuclear cells are not significantly affected by LBH589 + TKI, showing the specificity of this treatment regimen. Immunoblot analyses show that LBH589 + TKI induce apoptosis via degradation of FLT3-ITD and its prosurvival target STAT5. Previously, we showed the LBH589-induced proteasomal degradation of FLT3-ITD. Here, we show that activated caspase-3 also contributes to the degradation of FLT3-ITD and that STAT5 is a direct target of this protease. Our data strongly emphasize HDACi/TKI drug combinations as promising modality for the treatment of FLT3-ITD-positive AMLs., (©2012 AACR.)

Tissue engineering is an attractive approach to heal bony defects. However, three-dimensional cell-scaffold constructs display uneven oxygen supply resulting in inhomogeneous tissue quality. We assessed different strategies to improve oxygen supply in vitro. Scaffolds with differing inner surface were seeded with preosteoblastic cells and cultivated either statically or in perfusion bioreactors. Oxygen concentration and pH were measured in the center of the scaffolds. An inductive feedback mechanism was build to increase bioreactor pump speed according to the oxygen concentrations measured within the scaffolds. While pH remained stable, oxygen concentration decreased significantly under static conditions within the cell-seeded scaffolds. Reducing the scaffolds' inner surface as well as increasing perfusion speeds in bioreactors resulted in improved oxygen supply. We conclude that improving oxygen supply to three dimensional culture systems for bone tissue engineering is feasible in an automated manner. Culture conditions have to be adapted to each cell-scaffold system individually.

Fractures to the proximal fifth metatarsal bone are among the most frequent injuries to the foot. Various classifications intend to distinguish different fracture entities in regard to prognosis and treatment. The most commonly used classification by Lawrence and Botte delineates three fracture zones and gives treatment recommendations based on retrospective case series. Aim of our study was to critically review the literature and reevaluate the classification and treatment recommendations based on the highest level of evidence available. We performed a systematic literature search in Medline, Embase and Cochrane library and identified six prospective trials either comparing the same treatment for different fracture entities or different treatment strategies for the same fracture entity. The studies reveal that all "tuberosity avulsion fractures" (Zone 1, according to Lawrence and Botte) heal well using functional treatment. Even multifragmentary, displaced and intraarticular fractures in Zone 1 give comparable good results. Treatment with a short leg cast leads to a significant delay in return to preinjury level when compared to functional treatment. "Jones' fractures" (Zone 2) also demonstrate good to excellent results and complete bone healing when treated functionally. In contrast, "diaphyseal stress fractures" (Zone 3) at the distal limit of the fourth-fifth intermetatarsal articulation and just distally feature a significantly higher rate of treatment failure when treated non-operatively in a non-weight bearing short leg cast. Early intramedullary screw fixation leads to a significantly shorter time to bone healing and return to sport. In conclusion, acute fractures to the proximal fifth metatarsal bone should be classified into two entities only: First, metaphyseal fractures not extending beyond the distal end of the fourth-fifth intermetatarsal articulation, as these fractures, regardless the number of fragments, displacement and intraarticular involvement, should be treated functionally. Second, meta-diaphyseal fractures located at the distal end of the fourth-fifth intermetatarsal articulation or just distally, as these fractures require early intramedullary screw fixation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human mesenchymal stem cells (hMSCs) are regularly cultured and characterised under normoxic (21% O(2)) conditions, although the physiological oxygen tension in the stem cell niche is known to be as low as 1-2%. Oxygen itself is an important signalling molecule, but the distinct impact on various stem cell characteristics is still unclear. Therefore, the aim of this study was to evaluate the influence of oxygen concentration on the hMSC subpopulation composition, cell morphology and migration on different surfaces (polystyrene, collagen I, fibronectin, laminin) as well as on the expression of integrin receptors. Bone marrow-derived hMSCs were cultured either in normoxic (21% O(2)) or hypoxic (2% O(2)) conditions. The hMSC subpopulations were assessed by aspect ratio and cell area. Hypoxia promoted a more homogeneous cell population with a significantly higher fraction of rapidly self-renewing cells which are believed to be the true stem cells. Under hypoxic conditions hMSC volume and height were significantly decreased on all surfaces as measured by white light confocal microscopy. Furthermore, low oxygen tension led to a significant increase in cell velocity and Euclidian distance on all matrixes, which was evaluated by time-lapse microscopy. With regard to cell-matrix contacts, expression of several integrin subunits was evaluated by semi-quantitative RT-PCR. Increased expression of the subunits α(1), α(3), α(5,) α(6), α(11), α(v), β(1) and β(3) was observed in hypoxic conditions, while α(2) was higher expressed in normoxic cultured hMSCs. Taken together, our results indicate that hypoxic conditions promote stemness and migration of hMSC along with altering their integrin expression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A 63-year-old patient suffering from diabetes mellitus and arterial occlusive disease sustained a displaced fracture of the upper ankle joint. The fracture was treated by open reduction and internal fixation (ORIF) but 6 months later a delayed infection developed. Partial implant removal and a single lavage were performed. With persistent signs of infection full implant removal and subsequently debridement and lavage were carried out 3.5 months later followed by arthrodesis of the upper ankle joint. The arbitration board decided that the treatment applied after diagnosing the delayed infection was not sufficient which led to a delay in appropriate treatment. However, whether the arthrodesis of the upper ankle joint could have been prevented could not be proven.

The use of seeded scaffolds in regenerative medicine is limited by the low survival of transplanted mesenchymal stem cells (MSC). Current approaches aim at improving cell viability but require an adequate long-term detection of the transplanted cells. Unfortunately, commonly performed labeling techniques have not been validated for this purpose, and studies often reveal inconclusive results. Consequently, we intended to identify the most suitable method for long-term detection of human MSC (hMSC) in vitro and in vivo. hMSC were labeled using the vital stainings PKH26 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as well as enhanced green fluorescent protein (eGFP) transduction. Metabolic activity and relative fluorescence intensity (RFI) were quantified in vitro over 21 days at 8 time points using standardized semi-automated microscopy and flow cytometry. In vivo, cell seeded scaffolds were subcutaneously implanted in nude mice, and RFI was analyzed over 42 days at 5 time points. In vitro, PKH26 and CFDA-SE significantly reduced metabolic activity. RFI of both stainings significantly decreased after 1 day and further faded to <1% after 7 days. In contrast, labeling with eGFP showed no metabolic effect on hMSC, and no significant reduction of RFI over the total period of 21 days. In vivo, RFI of eGFP labeled cells reached a plateau phase after 21 days and displayed a 3.8-fold higher RFI compared with PKH26 and CFDA-SE on day 42 evaluated in 280 field of views per scaffold using three scaffolds for each labeling technique and time point. We conclude that PKH26 and CFDA-SE are unsuitable for long-term detection of hMSC. eGFP transduction, in turn, allows long-term detection of hMSC in vitro and in vivo. Our results suggest that eGFP is currently the best option among the fluorescent labeling techniques to follow the fate of transplanted hMSC.

Acute ankle injuries are among the most common injuries in emergency departments. However, there are still no standardized examination procedures or evidence-based treatment. Therefore, the aim of this study was to systematically search the current literature, classify the evidence, and develop an algorithm for the diagnosis and treatment of acute ankle injuries. We systematically searched PubMed and the Cochrane Database for randomized controlled trials, meta-analyses, systematic reviews or, if applicable, observational studies and classified them according to their level of evidence. According to the currently available literature, the following recommendations have been formulated: i) the Ottawa Ankle/Foot Rule should be applied in order to rule out fractures; ii) physical examination is sufficient for diagnosing injuries to the lateral ligament complex; iii) classification into stable and unstable injuries is applicable and of clinical importance; iv) the squeeze-, crossed leg- and external rotation test are indicative for injuries of the syndesmosis; v) magnetic resonance imaging is recommended to verify injuries of the syndesmosis; vi) stable ankle sprains have a good prognosis while for unstable ankle sprains, conservative treatment is at least as effective as operative treatment without the related possible complications; vii) early functional treatment leads to the fastest recovery and the least rate of reinjury; viii) supervised rehabilitation reduces residual symptoms and re-injuries. Taken these recommendations into account, we present an applicable and evidence-based, step by step, decision pathway for the diagnosis and treatment of acute ankle injuries, which can be implemented in any emergency department or doctor's practice. It provides quality assurance for the patient and promotes confidence in the attending physician.

Purpose: Bupivacaine, ropivacaine, and morphine are commonly administered intraarticularly after anterior cruciate ligament (ACL) reconstruction. However, their effects on human tendon stem/progenitor cells (TSPC) have not been studied. Therefore, this study investigates the cytotoxicity of these analgetics on TSPC., Methods: Cells were isolated from human hamstring grafts of 3 female (age 15, 16 and 59) and 2 male patients (age 16 and 47). Cells were incubated using 0.5% bupivacaine, 0.5/0.75% ropivacaine, and 0.025% morphine. Cell viability was assessed after 0.5, 2, and 6 h using live/dead assay. Metabolic activity and apoptosis were measured by WST- and Annexin-V-FACS-assay after 2 h., Results: Cell viability remained unchanged after 0.5 h in all groups, while treatment with bupivacaine and 0.5/0.75% ropivacaine resulted in a complete cell loss after 6 h. Contrarily, morphine showed no cytotoxic effect. Cell viability and metabolism were significantly reduced after treatment with bupivacaine (22.1; 8.3%) and 0.75% ropivacaine (56.5; 23.8%), while 0.5% ropivacaine and morphine showed no significant difference compared with controls. Apoptosis was significantly induced after incubation with bupivacaine (58.1%) and 0.75% ropivacaine (26.2%), whereas 0.5% ropivacaine only led to a slight induction compared with morphine and controls., Conclusions: Clinically administered concentrations of bupivacaine (0.5%) and ropivacaine (0.75%) have a significant cytotoxic effect on human TSPC in vitro, while ropivacaine in a concentration of 0.5% has a mild but not significant effect on apoptosis and cell metabolism. In contrast, morphine does not affect cell survival, metabolism, or apoptosis. Knowing that morphine provides comparable to even prolonged pain reduction after ACL reconstruction, the presented in vitro study suggests morphine as a potentially less toxic analgetic drug for intraarticular application in clinical practice.

Assessment of cell fate is indispensable to evaluate cell-based therapies in regenerative medicine. Therefore, a widely used technique is fluorescence labeling. A major problem still is the standardized, noninvasive, and reliable quantification of fluorescence intensity of adherent cell populations on single-cell level, since total fluorescence intensity must be correlated to the cell number. Consequently, the aim of the present study was to produce and validate an open-source-based algorithm, capable of measuring the total fluorescence intensity of cell populations and assessing the total cell number in phase-contrast images. To verify the algorithms' capacity to assess fluorescence intensity, human mesenchymal stem cells were transduced to stably express enhanced green fluorescent protein and results produced by the algorithm were compared to flow cytometry analysis. No significant differences could be observed at any time (p ≥ 0.443). For validation of the algorithm for cell counting in phase-contrast images, adherent human mesenchymal stem cells were manually counted and compared to results produced by the algorithm (correlation coefficient [CC] r = 0.975), nuclei staining (CC r = 0.997), and hemocytometer (CC r = 0.629). We conclude that applying the developed algorithm in routine practice allows robust, fast, and reproducible assessment of fluorescence intensity and cell numbers in simple large-scale microscopy. The method is easy to perform and open source based.

Introduction: The physical examination of the knee in cases of suspected meniscal tears serves to increase the probability of a correct diagnosis. Although there is a large variety of functional tests, the quality of each diagnostic test is controversially discussed., Materials and Methods: Through a systematic literature search in Medline and the Cochrane Database two reviewers independently screened publications, evaluated each study for methodological quality and categorized them into levels of evidence (CEBM). Sensitivity, specificity, positive and negative predicted value, as well as positive and negative likelihood ratio (LR+/LR-) values were calculated in order to render the quality threshold of the physical examination in meniscus impairment., Results: The Thessaly test (sensitivity: 91%, specificity: 97%, PPV: 97%, NPV: 91% LR+: 31.1, LR-: 0.1) revealed the highest test quality. Limited quality was shown for the Mc Murray test (sensitivity: 51%, specificity: 78%, PPV: 70%, NPV: 61%, LR+: 2.3, LR-: 0.6), "joint line tenderness" (sensitivity: 64%, specificity: 61%, PPV: 62%, NPV: 63%, LR+: 1.6, LR-: 0.6), the Apley-Grinding test (sensitivity: 38%, specificity: 84%, PPV: 71%, NPV: 58%, LR+: 2.4, LR-: 0.7) and the Ege test (sensitivity: 66%, specificity: 86%, PPV: 83%, NPV: 72%, LR+: 4.7, LR-: 0.4). Evidence for Steinman's test, Bragard's test and the meniscal signs of Böhler or Payr could not be tested., Conclusion: Meniscal injury can be detected by several functional tests. Using the Thessaly test can improve the physical examination by means of probability of the correct diagnosis, but the results are based on a single study. In patients with ambiguous findings in the physical examination or with suspected combined injury, further diagnostic procedures such as magnetic resonance imaging are necessary to confirm the diagnosis. In clinically certain cases the use of additional diagnostic imaging procedures should be avoided as other authors have shown that with few exceptions this has no influence on the therapy.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts is a prerequisite for subsequent bone formation. Numerous studies have explored osteogenic differentiation under standard tissue culture conditions, which usually employ 21% of oxygen. However, bone precursor cells such as hMSCs reside in stem cell niches of low-oxygen atmospheres. Furthermore, they are subjected to low oxygen concentrations when cultured on three-dimensional scaffolds in vitro, and even more so after transplantation when vascularization has yet to be established. Similarly, hMSCs are exposed to low oxygen in the fracture microenvironment following bony injury. Recent studies revealed that hypoxic preconditioning improves cellular engraftment and survival in low-oxygen atmospheres. In our study we investigated the osteogenic differentiation potential of hMSCs under 2% O(2) (hypoxia) in comparison to a standard tissue culture oxygen atmosphere of 21% (normoxia). We assessed the osteogenic differentiation of hMSCs following hypoxic preconditioning to address whether this pretreatment is beneficial for subsequent differentiation processes as well. To validate our findings we carefully characterized the extent of hypoxia exerted and its effect on cell survival and proliferation. We found that hMSCs proliferate better if cultured under 2% of oxygen. We confirmed that osteogenic differentiation of hMSCs is indeed inhibited if osteogenic induction is carried out under constant hypoxia. Finally, we showed for the first time that hypoxic preconditioning of hMSCs prior to osteogenic induction restores osteogenic differentiation of hMSCs under hypoxic conditions. Collectively, our results indicate that maintaining constant levels of oxygen improves the osteogenic potential of hMSCs and suggest that low oxygen concentrations may preserve the stemness of hMSCs. In addition, our data support the hypothesis that if low-oxygen atmospheres are expected at the site of implantation, hypoxic pretreatment may be beneficial for the cells' subsequent in vivo performance.

The distribution of unpaired electron spin over all regions of the organic ligands was extracted from the large positive and negative 1H and 13C NMR paramagnetic shifts of the title complexes. Owing to benevolent line broadening and to very high sensitivities of approximately 254,000 and approximately 201,000 ppm/(unpaired electron spin) for Co(II) and Ni(II), respectively, at 298 K in these pseudotetrahedral bis(N,N'-chelates), spin transmission through the sigma- (and orthogonal pi)-bonding system of the ligands could be traced from the chelate ring over five to nine sigma bonds. Most of those "experimental" spin densities DeltarhoN (situated at the observed nuclei) agree reasonably well with quantum chemical DeltarhoDFT (DFT = density functional theory) values and provide an unsurpassed number of benchmark values for the quality of certain types of modern density functionals. The extraction of DeltarhoN became possible through the unequivocal separation of the nuclear Fermi contact shift components from the metal-centered pseudocontact shifts, which are proportional to the anisotropy Deltachi of the magnetic susceptibility: Experimental Deltachi values were obtained in solution from measured deuterium quadrupole splittings in the 2H NMR spectra of two deuterated model complexes and were found to be nonlinear functions of the reciprocal temperature. This provided the reliable basis for predicting metal-centered pseudocontact shifts for any position of a topologically well-defined ligand at varying temperatures. The related ligand-centered pseudocontact shifts were sought by using the criterion of their expected nonlinear dependence on the reciprocal temperature. However, their contributions could not be differentiated from other small effects close to the metal center; otherwise, they appeared to be smaller than the experimental uncertainties. The free activation energy of N-aryl rotation past a vicinal tert-butyl substituent in the Ni(II) vinamidine bis(N,N'-chelates) is DeltaG++(+74 degrees C) approximately 17.0 kcal/mol and past a vicinal methyl group DeltaG++(-6 degrees C) approximately 13.1 kcal/mol.

Purpose: We assessed dynamic contrast enhanced magnetic resonance imaging as a biological marker of in vivo changes in microcirculation in the prostatic suburethral region., Materials and Methods: A total of 12 male beagle dogs with spontaneous benign prostatic hyperplasia were randomly allocated to 1 control group and 1 finasteride (Merck and Co., Whitehouse Station, New Jersey) treated group. Two baseline dynamic contrast enhanced magnetic resonance imaging examinations and 3 followups were performed to assess prostate microcirculation. Treatment duration was 3 months. The pharmacokinetic parameters evaluated in prostatic suburethral areas were the maximum enhancement ratio in AU, time to maximum signal enhancement in minutes, amplitude in AU and the exchange rate constant in minutes(-1)., Results: After completion of the therapeutic regimen time to maximum signal enhancement was significantly longer in the finasteride group than in controls (p < 0.01). Amplitude and the exchange rate constant decreased 39% and 34%, respectively, in the finasteride group at the end of treatment, which significantly differed from results in the control group (p < 0.05)., Conclusions: Dynamic contrast enhanced magnetic resonance imaging is capable of noninvasively assessing the prostatic microcirculation changes induced by finasteride. Pharmacokinetic parameters show considerable promise to be biomarkers for the development of benign prostatic hyperplasia drugs such as 5alpha-reductase inhibitors by the in vivo monitoring of microvascular changes. A relevant clinical application could be the pretreatment assessment of finasteride effectiveness to decrease perioperative bleeding at transurethral prostate resection and in treatment for hematuria.

We sought to assess the possibility of using pharmacokinetic parameters as a predictor of response to benign prostatic hyperplasia (BPH) pharmacotherapy via a randomized, placebo-controlled, animal preclinical trial using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Twelve male beagles with BPH were enrolled in a preclinical experimental drug trial and divided into two randomized groups with six beagles each: one drug (finasteride) group and one placebo (control) group. Two baseline MRI examinations and three follow-ups during treatment were performed on a clinical 1.5-T MRI system using axial T1- and T2-weighted magnetic resonance images for prostate volume measurement and DCE-MRI for the assessment of prostate microcirculation. A total of 0.2 mmol/kg body weight of the Gd-based contrast agent was administered with an injection rate of 0.2 ml/s. The pharmacokinetic parameters, maximum enhancement ratio (MER), transfer constant and rate constant, were assessed to characterize the microcirculation in the parenchymal zone. The time-signal intensity curve from the external iliac artery was used as the arterial input function. The correlation between baseline evaluations (prostate volume and pharmacokinetic parameters) and therapy-induced prostate volume changes under finasteride treatment were assessed. The changes in prostate volume at the end of the trial exhibited a significant linear correlation to the initial parenchymal MER (P < .02) in the finasteride group. Larger prostate volume reductions coincided with smaller initial parenchymal MER. These findings show considerable promise of using parenchymal MER as a predictor of response to BPH pharmacotherapy with finasteride.

Objectives: We sought to evaluate the capabilities of different magnetic resonance imaging (MRI)-based methodologies for measuring prostate volume., Materials and Methods: Twenty-four male beagles with benign prostatic hyperplasia were enrolled in a drug trial and imaged at 5 time points. A total of 120 prostate volumes were determined by MRI-based semiautomated segmentation. For planimetric assessment, 8 diameter locations were determined in the axial and coronal plane of the MRI slice with maximum extension of the prostate. Thirteen calculation models based on these diameters were determined by comparison to the reference volume and evaluated during treatment., Results: The segmented MRI prostate volume significantly correlated with post necropsy volume. The best diameter-based model also worked very well for monitoring prostate volume of dogs under treatment., Conclusions: MRI-based segmentation is highly accurate in assessing prostate volume. Diameter-based measurements are closely correlated to the segmented prostate volume and are feasible to monitor therapy.

In a prospective, randomised, double-masked, parallel-group, multi-centre study, 210 patients with primary open angle glaucoma or ocular hypertension were enrolled of whom 167 (timolol 0.1% gel 82, timolol 0.5% 85) completed the study as per protocol. The change in intraocular pressure between baseline and week 12 in the worse eye ('at trough') was 6.3 (SD 3.3) mm Hg on timolol 0.1% gel and 7.0 (2.9) mm Hg on timolol 0.5%; this difference was not statistically significant (p = 0.19). The difference between the two study groups in the change of intraocular pressure from baseline was 0.62 mm Hg; the 90% CI of -0.09 to +1.33 mm Hg was within the pre-specified limits of -1.5 to +1.5 mm Hg demonstrating equivalence between timolol 0.1% gel and timolol 0.5%. The plasma levels of timolol (ng/ml) at 12 weeks in the timolol 0.1% gel group were significantly less than that with timolol 0.5% both before instillation (mean 0.057, SD 0.131 and mean 0.470, SD 0.519 respectively, p = 0.025) and after instillation (mean 0.552, SD 0.992 and mean 2.473, SD 1.780 respectively, p = 0.008). Both treatments were well tolerated with no statistically significant difference between the groups in the occurrence of ocular or systemic adverse events., (Copyright 2002 S. Karger AG, Basel)